Macrophage-Specific E3 Ubiquitin Ligase TRIM31 Reduces Atherosclerotic Plaque Formation by Targeting LOX-1

Atherosclerosis is a chronic inflammatory disease marked by lipid accumulation and immune cell infiltration in arterial walls. Macrophages contribute by internalizing oxidized low-density lipoprotein, forming foam cells, and driving inflammation. The ubiquitin-proteasome system regulates immune and inflammatory responses in atherosclerosis. This study investigated the protective role of TRIM31 (tripartite motif-containing 31), an E3 ubiquitin ligase, in macrophage lipid metabolism and inflammation through selective regulation of LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1).

Transcriptomic profiling, macrophage-specific Trim31 knockout (Trim31^fl/flLyz2^cre) and overexpression (Trim31^Lyz2-KI) mice, and Lox-1 knockout (Lox-1^-/-) models were used to examine the impact of TRIM31 in vivo (n=8 per group). TRIM31 substrates were identified using single-cell RNA sequencing of atherosclerotic aortas and proteomic/immunoprecipitation-mass spectrometry analyses. Functional assays were performed in both mouse and human macrophages (n=5-6 per group). Ubiquitination mechanisms were clarified through immunoprecipitation and site-directed mutagenesis. Rescue experiments involved Lox-1 knockdown or reconstitution with wild-type and lysine 12 to arginine variant (K12R) Lox-1 and Trim31 overexpression in Lox-1^-/- or Apoe^-/-Lox-1^-/- mice to evaluate the functional importance of Lox-1 ubiquitination in vivo (n=8 per group) and in vitro (n=5 per group).

TRIM31 was selectively upregulated in macrophages under oxidized low-density lipoprotein stimulation and in atherosclerosis plaques. Trim31 deficiency exacerbated plaque burden, foam cell formation, and inflammatory signaling (n=8 per group). Single-cell analysis revealed enrichment of lipid transport and inflammatory pathways in Trim31-deficient plaques. LOX-1 was identified as a key TRIM31 substrate. TRIM31 promoted K48-linked ubiquitination of LOX-1 at lysine 12, facilitating its degradation. The atheroprotective effects of Trim31 were abolished in Lox-1^-/- or K12R-variant rescue models. The TRIM31-LOX-1 axis was also confirmed by human macrophages in regulating lipid uptake and inflammation.

TRIM31, an inducible, macrophage-enriched protective factor in atherosclerosis, restricts foam cell formation and inflammation by targeting LOX-1 for proteasomal degradation. These findings position TRIM31 as a promising therapeutic target for macrophage-driven atherogenesis.