A Chemical Proteomic Map of Heme-Protein Interactions

Abstract

Heme is an essential cofactor for many human proteins as well as the primary transporter of oxygen in blood. Recent studies have also established heme as a signaling molecule, imparting its effects through binding with protein partners rather than through reactivity of its metal center. However, the comprehensive annotation of such heme-binding proteins in the human proteome remains incomplete. Here, we describe a strategy which utilizes a heme-based photoaffinity probe integrated with quantitative proteomics to map heme-protein interactions across the proteome. In these studies, we identified 350+ unique heme-protein interactions, the vast majority of which were heretofore unknown and consist of targets from diverse functional classes, including transporters, receptors, enzymes, transcription factors, and chaperones. Among these proteins is the immune-related interleukin receptor-associated kinase 1 (IRAK1), where we provide preliminary evidence that heme agonizes its catalytic activity. Our findings should improve the current understanding of heme's regulation as well as its signaling functions and facilitate new insights of its roles in human disease.

Figure 1.

Chemical proteomic approach to map heme–protein interactions. (a) Schematic workflow using heme-based photoaffinity probe (HPAP) to identify heme-binding proteins. (b) Structure of HPAP, hemin, and FF-control. (c) Gel-based profiling of HPAP-protein interactions in HEK293T lysates. Lysates were incubated with increasing concentrations of HPAP, photo-cross-linked, then conjugated to a TAMRA-azide tag by CuAAC “click” chemistry prior to fluorescence scanning. (d) Competitive blockade of HPAP protein labeling by free hemin.

Figure 2.

Confirmation of HPAP and heme interactions with known heme-binding proteins. FLAG-tagged proteins were recombinantly expressed in HEK293T cells, lysed, and cotreated with HPAP and increasing concentrations of free hemin prior to visualization. Full images are shown in Figure S3.

Figure 3.

MS-based profiling of HPAP-protein interactions in human cell lysates. (a) Scatter plots comparing heme-binding proteins in HEK293T, K562, and PBMC lysates. Heme-binding proteins (black) are defined as exhibiting ≥4-fold enrichment (x-axis) by HPAP over FF-control (25 μM) and competed (y-axis) ≥3-fold with excess hemin (100 μM), indicated by red dotted lines. (b) Target overlap between HEK293T (red), K562 (green), and PBMC (purple) cells. (c) Protein class distribution of targets. (d) Top ten biochemical pathways enriched across all targets.

Figure 4.

Confirmation of newly identified heme-binding proteins. FLAG-tagged proteins were recombinantly expressed in HEK293T cells, lysed, and cotreated with HPAP and increasing concentrations of free hemin prior to visualization. Full images are shown in Figure S5.

Figure 5.

Characterization of newly identified heme-binding proteins. (a) Quantitation of ATP consumption by IRAK1 treated with heme or DMSO via ADP-Glo assay. (b) Steady state kinetics of IRAK1 to determine the K_m and V_max in the presence of hemin. (c) Immunoblot analysis of IRAK1phosphorylation in HEK293T cell lysates treated with increasing concentrations of hemin followed by SDS-PAGE separation using a phosphor-binding gel matrix. (d) Quantitation of pIRAK1 immunoblotting (mean ± SD, n = 4; Figure S6).