School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.
School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Metallomics. 2023 Mar 6;15(3). doi: 10.1093/mtomcs/mfad004.
Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.
血红素 b（铁原卟啉 IX）作为金属辅因子和信号分子在生物学中发挥着重要作用。然而，血红素信号的靶标以及介导血红素从合成或摄取部位到血红素依赖或调节蛋白的蛋白质交换的网络还知之甚少。在此，我们描述了一种基于定量质谱（MS）的化学生物学策略，用于鉴定人胚肾 HEK293 细胞中可能与血红素信号转导和运输相关的可交换不稳定血红蛋白。该策略涉及用血红素生物合成抑制剂琥珀酰丙酮（SA）耗尽内源性血红素，使假定的血红素结合蛋白处于脱辅基状态，然后使用血红素琼脂糖树脂捕获这些蛋白，最后通过 MS 进行洗脱和鉴定。通过仅鉴定那些以 SA 依赖的方式与血红素琼脂糖高特异性相互作用的蛋白，而不是与对照珠状琼脂糖相互作用的蛋白，我们扩展了可能参与结合和缓冲不稳定血红素或血红素信号靶标的蛋白的数量和类别。值得注意的是，这些蛋白包括涉及染色质重塑、DNA 损伤反应、RNA 剪接、细胞骨架组织和囊泡运输的蛋白，其中许多蛋白已通过最近发表的互补研究与血红素相关。总之，这些结果为血红素在从基因组完整性到蛋白质运输等一系列扩展的细胞过程中发挥作用提供了支持。
