QUANTITATIVE PROTEOMIC REVEALS HEME OXYGENASE-1  LOCALIZATION TO CELL-SURFACE LIPID RAFTS AND ITS  ASSOCIATION WITH FERROPORTIN IN IRON-LOADED  MACROPHAGES
​bioRxiv preprint doi: https://doi.org/10.64898/2026.09.14.750975

RUNNING TITLE: Hmox1 co-localizes with ferroportin at the cell surface of iron overloaded macrophages
Alexandra WILLEMETZ1,2*, Anne AURIAC3*, Christian FEDERICI4, Lorenne ROBERT5, Marjorie  LEDUC4, Luc CAMOIN6 and François CANONNE-HERGAUX7#
AFFILIATION: 1 Université de Paris Cité, F-75015, Paris, France ; 2INSERM, U1149, Centre de Recherche sur 10 l'Inflammation, 75018, Paris, France; 3 INSERM UMR 1043, CNRS UMR 5282, Université de Toulouse, UPS, 11 Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France; IRSD, Université de Toulouse, 12 INSERM, INRA, INP-ENVT, Université de Toulouse 3 Paul Sabatier, Toulouse, France.; 4Proteom'IC facility, 13 Institut Cochin, CNRS, INSERM, Université Paris Cité, PARIS, France. 5INSERM Toulouse NeuroImaging 14 Center (ToNIC), Inserm, University of Toulouse (UPS), Toulouse, France ; 6Aix Marseille Univ, CNRS, INSERM, 15 Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille Proteomics, 16 Marseille, France ; 7Université de la Réunion, INSERM, UMR 1188 Diabète Athérothrombose Thérapies 17 Réunion Océan Indien (DéTROI), Saint-Pierre, France.

Abstract

Background: Macrophages play a central role in systemic iron homeostasis by recycling iron from senescent erythrocytes through the only known cellular iron exporter, ferroportin (Fpn). Previous studies have shown that Fpn localizes to membrane microdomains, or lipid rafts, which are important for its function and regulation by hepcidin. Iron strongly promotes the enrichment of Fpn in lipid rafts. However, the proteomic composition of these domains during iron overload remains largely unexplored.
Objective: This study aimed to characterize iron-induced changes in the macrophage lipid raft proteome in order to identify potential functional partners of Fpn and better define the cellular mechanisms involved in the response to iron overload.
Methods: Quantitative iTRAQ-based proteomic analysis was performed on detergent-resistant membranes (DRMs) isolated by iodixanol density-gradient ultracentrifugation from J774a.1 macrophages treated with iron-NTA (FeNTA). Key proteomic observations were further validated in both J774a.1 cells and bone marrow-derived macrophages (BMDMs) using western blot, cell-surface biotinylation, and confocal immunofluorescence microscopy. The regulatory role of the transcription factor Nrf2 was also assessed using Nrf2-knockout mouse models.
Results: In addition of Fpn, our lipid raft proteome identified 79 proteins significatively upregulated upon iron treatment, including, antioxidant proteins like the peroxiredoxin-1 (Prdx1), the glucose-6-phosphate dehydrogenase X-linked (G6pdx), as well as heme oxygenase-1 (Hmox1). Bioinformatic and functional analyses indicated that this response is associated with nuclear translocation of Nrf2, which is required for the transcriptional induction of Fpn and Hmox1. Whereas Fpn was detected in lipid rafts under basal conditions, Hmox1 was recruited to these domains only after iron or heme treatment. Confocal microscopy and cell-surface biotinylation assays confirmed that Hmox1 and Fpn colocalize at the plasma membrane, particularly within caveolae, specialized invaginated lipid raft domains.
Conclusions: Iron or heme overload induces a major remodeling of the macrophage membrane, leading to the formation of specialized lipid raft platforms enriched in antioxidant and iron handling proteins. The coordinated localization of Hmox1 and Fpn at the cell surface suggests a coupled mechanism linking heme catabolism to iron export. This organization may help limit intracellular iron accumulation and protect the plasma membrane from iron-mediated lipid peroxidation

doi: https://doi.org/10.64898/2026.09.14.750975