Nuñez Raymundo, Sidlowski Paul F W, Steen Erica A, Wynia-Smith Sarah L, Sprague Daniel J, Keyes Robert F, Smith Brian C
Department of Biochemistry, Program in Chemical Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
ACS Chem Biol. 2024 Dec 20;19(12):2418-2428. doi: 10.1021/acschembio.4c00248. Epub 2024 Nov 18.
Histone lysine lactylation (Kla) regulates inflammatory gene expression in activated macrophages and mediates the polarization of inflammatory (M1) to reparative (M2) macrophages. However, the molecular mechanisms and key protein players involved in Kla-mediated transcriptional changes are unknown. As Kla is structurally similar to lysine acetylation (Kac), which is bound by bromodomains, we hypothesized that bromodomain-containing proteins bind histone Kla. Here, we screened 28 recombinantly expressed bromodomains for binding to histone Kla peptides via AlphaScreen assays. TRIM33 was the sole bromodomain tested that bound histone Kla peptides. TRIM33 attenuates inflammatory genes during late-stage macrophage activation; thus, TRIM33 provides a potential link between histone Kla and macrophage polarization. Orthogonal biophysical techniques, including isothermal titration calorimetry and protein-detected nuclear magnetic resonance, confirmed the submicromolar binding affinity of the TRIM33 bromodomain to both Kla and Kac histone post-translational modifications. Sequence alignments of human bromodomains revealed a unique glutamic acid residue within the TRIM33 binding pocket that we found confers TRIM33 specificity for binding Kla compared with other bromodomains. Molecular modeling of interactions of Kla with the TRIM33 bromodomain binding pocket and site-directed mutagenesis of glutamic acid confirmed the critical role of this residue in the selective recognition of Kla by TRIM33. Collectively, our findings implicate TRIM33, a bromodomain-containing protein, as a novel reader of histone Kla, potentially bridging the gap between histone Kla and macrophage polarization. This study enhances our understanding of the regulatory role of histone Kla in macrophage-mediated inflammation and offers insights into the underlying structural and biophysical mechanisms.
组蛋白赖氨酸乳酰化（Kla）调节活化巨噬细胞中的炎症基因表达，并介导炎症性（M1）巨噬细胞向修复性（M2）巨噬细胞的极化。然而，Kla介导的转录变化所涉及的分子机制和关键蛋白尚不清楚。由于Kla在结构上与赖氨酸乙酰化（Kac）相似，而Kac可被溴结构域结合，我们推测含溴结构域的蛋白可结合组蛋白Kla。在此，我们通过AlphaScreen分析筛选了28种重组表达的溴结构域与组蛋白Kla肽段的结合情况。TRIM33是唯一被检测到能结合组蛋白Kla肽段的溴结构域。TRIM33在巨噬细胞活化后期减弱炎症基因表达；因此，TRIM33在组蛋白Kla和巨噬细胞极化之间提供了潜在联系。包括等温滴定量热法和蛋白质检测核磁共振在内的正交生物物理技术证实了TRIM33溴结构域与Kla和Kac组蛋白翻译后修饰的亚微摩尔结合亲和力。人类溴结构域的序列比对显示，TRIM33结合口袋内有一个独特的谷氨酸残基，我们发现该残基赋予TRIM33与其他溴结构域相比对Kla结合的特异性。Kla与TRIM33溴结构域结合口袋相互作用的分子模型以及谷氨酸的定点诱变证实了该残基在TRIM33对Kla的选择性识别中的关键作用。总的来说，我们的研究结果表明，含溴结构域的蛋白TRIM33是组蛋白Kla的新型识别蛋白，可能弥合组蛋白Kla与巨噬细胞极化之间的差距。这项研究增进了我们对组蛋白Kla在巨噬细胞介导的炎症中的调节作用的理解，并为潜在的结构和生物物理机制提供了见解。
