WILLEMETZ, A. | bioRxiv Science

bioRxiv2026

QUANTITATIVE PROTEOMIC REVEALS HEME OXYGENASE-1 LOCALIZATION TO CELL-SURFACE LIPID RAFTS AND ITS ASSOCIATION WITH FERROPORTIN IN IRON-LOADED MACROPHAGES

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.

cell biology↗

bioRxiv2023

NEW INSIGHTS INTO THE HEPATIC IRON PHENOTYPE OF BMP6 KNOCKOUT MICE

ObjectiveBmp6 knockout (KO) mice progressively accumulate a significant amount of iron in their liver as they age due to a defect in hepcidin (Hamp) expression and an upregulation of the iron exporter ferroportin (Fpn). In this study, we conducted a comprehensive investigation of the hepatic iron overload phenotype, with specific emphasis on the cellular and subcellular localization of Fpn in Bmp6 KO mice. Materials and MethodsLivers obtained from Bmp6 knockout (KO) mice at different developmental stages were utilized for the quantification of iron content, investigation of iron distribution, histological analysis, histoimmunofluorescence assays performed on paraffin-embedded sections, confocal microscopy examinations, subcellular membrane fractionation, and western blot analysis. ResultsIn Bmp6 KO livers, iron overload increased with age and was not homogeneous, with certain hepatic lobes and specific areas in liver sections showing more pronounced iron accumulation. In young mice, iron accumulated mostly in the centrilobular zone where low Fpn expression was observed. Fpn was strongly detected in periportal Kupffer cells and at the apical membrane of periportal hepatocytes lining the sinusoidal capillaries. The zonal distribution of iron tended to disappear with age in strongly iron-overloaded areas, with the appearance of large cellular aggregates strongly positive for Fpn, iron, and ceroid/lipofuscin. At the subcellular level, hepatic Fpn seemed to concentrate in specific cell surface compartments and was enriched in a lipid raft fraction. ConclusionsUnregulated expression of Fpn on the cell surface of periportal macrophages and hepatocytes results in centrilobular iron overload within hepatocytes. In areas of pronounced iron overload, Fpn expression is present in lipogranulomas, identified as aggregations of macrophages accumulating hemosiderin and ceroid/lipofuscin pigments. These lesions likely form due to the phagocytosis of sideronecrotic/ferroptotic hepatocytes by macrophages. In contrast to the duodenal form of Fpn, both splenic and hepatic Fpn demonstrated robust enrichment within lipid rafts. The observed variations in the subcellular localization of Fpn could play a significant role in influencing the transporters iron transport activity and/or its regulation by hepcidin.

pathology↗