Protein aggregation as a cellular response to oxidative stress induced by heme and iron

Abstract

Hemolytic diseases include a variety of conditions with diverse etiologies in which red blood cells are destroyed and large amounts of hemeproteins are released. Heme has been described as a potent proinflammatory molecule that is able to induce multiple innate immune responses, such as those triggered by TLR4 and the NLRP3 inflammasome, as well as necroptosis in macrophages. The mechanisms by which eukaryotic cells respond to the toxic effects induced by heme to maintain homeostasis are not fully understood, however. Here we describe a previously uncharacterized cellular response induced by heme: the formation of p62/SQTM1 aggregates containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). This action is part of a response driven by the transcription factor NRF2 to the excessive generation of reactive oxygen species induced by heme that results in the expression of genes involved in antioxidant responses, including p62/SQTM1. Furthermore, we show that heme degradation by HO-1 is required for ALIS formation, and that the free iron released on heme degradation is necessary and sufficient to induce ALIS. Moreover, ferritin, a key protein in iron metabolism, prevents excessive ALIS formation. Finally, in vivo, hemolysis promotes an increase in ALIS formation in target tissues. Our data unravel a poorly understood aspect of the cellular responses induced by heme that can be explored to better understand the effects of free heme and free iron during hemolytic diseases such as sickle cell disease, dengue fever, malaria, and sepsis.

Keywords: ALIS; autophagy; heme; iron; p62/SQSTM1.

Fig. 1.

Heme-induced ALIS do not depend on autophagy machinery. (A) Immunoblot analysis of LC3-I to LC3-II conversion of RAW GFP-LC3 cells stimulated with heme (30 or 100 μM) for 2 h or with rapamycin (50 μg/mL) for 2 h. Actin served as a loading control. (B) Fluorescence microscopy of RAW GFP-LC3 cells stimulated with heme (100 μM) for 6 h or with rapamycin (50 μg/mL) for 2 h. (Insets) Heme- and rapamycin-induced GFP-LC3⁺ structures. (C) Immunoblots for LC3-I to LC3-II conversion and p62 from extracts of WT and Atg5^−/− MEFs stimulated with heme (100 μM) for 6 h or with rapamycin (50 μg/mL) for 2 h. Actin served as a loading control. (D) Fluorescence microscopy of WT and Atg5^−/− MEFs stimulated with heme (100 μM) for 12 h. (E) Quantification of GFP-LC3⁺ dots in RAW GFP-LC3 cells stimulated with heme (30 or 100 μM) for 6, 12, or 24 h. (F) Quantification of GFP-LC3⁺ dots in RAW cells pretreated with vehicle or wortmannin (200 nM) and stimulated with heme (100 μM) for 12 or 24 h. (G) Quantification of GFP-LC3⁺ dots in WT and Atg5^−/− MEFs stimulated with heme (100 μM) for 12 or 24 h. (H) Quantification of p62⁺ dots in WT and Atg5^−/− MEFs stimulated with heme (100 μM) for 4, 6, 12, or 24 h. (I) Immunofluorescence images showing cellular distribution of p62 (red) and Ub (green) in BMDMs stimulated with heme (100 μM) for 6 h. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05; ns, not significant.

Fig. S1.

Heme induces LC3⁺ dots that colocalize with ALIS. (A and B) Immunofluorescence microscopy images of RAW GFP-LC3 cells stimulated with heme (100 μM) for 12 h or with puromycin (5 μg/mL) and stained for p62 (red) (A) or Ub (green) (B). (C and D) Immunofluorescence showing the colocalization of p62 (red) and Ub (green) in RAW cells (C) and in immortalized BMDMs (D) stimulated with heme 100 μM for 12 h. Nuclei were stained with DAPI (blue). (Scale bars: 5 μm.)

Fig. S2.

Indiction of ALIS by heme does not require TLR4 and MYD88. (A) RAW GFP-LC3 cells stimulated with heme 100 μM or LPS (100 ng/mL) for 12 h. (B–D) Immunofluorescence microscopy for p62 (red) and Ub (green) of BMDMs stimulated with heme 100 μM or LPS (100 ng/mL) for 6 h (B) and quantification of p62⁺ (C) and Ub⁺ dots (D) of BMDMs stimulated as in B. (E) Secretion of TNF in BMDMs from WT and TLR4^−/− mice stimulated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL), or heme (30, 50 or 100 μM) for 6 h. (F and G) Immunofluorescence images of WT (F) and TLR4^−/− (G) BMDMs stimulated with heme (100 μM), LPS (100 ng/mL), or Pam3CSK4 (200 ng/mL) for 6 h and stained for p62 (red) and Ub (green). (H and I) Quantification of p62⁺ (H) and Ub⁺ (I) dots in BMDMs from WT and TLR4^−/− mice stimulated as in A and B. (J and K) Immunofluorescence microscopy of WT (J) and Myd88^−/− (K) immortalized BMDMs stimulated with heme (100 μM), LPS (100 ng/mL), or Pam3CSK4 (200 ng/mL) for 6 h and stained for p62 (red) and Ub (green). (L) Quantification of p62⁺ (red) dots in WT and MyD88^−/− immortalized BMDMs stimulated with heme (100 μM) or LPS (100 ng/mL) for 6 h. (M) Quantification of p62⁺ dots in BMDMs left untreated or stimulated with 1, 100, or 1,000 ng/mL of LPS for 6 h in the presence or absence of heme (100 μM) for 6 h. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. S3.

MAPK and ROS are necessary for the formation of heme-induced ALIS. (A) Immunofluorescence microscopy of p62 (red) and Ub (green) in BMDMs pretreated with vehicle or ERK, p38, or JNK inhibitors and stimulated with heme (100 μM) or LPS (100 ng/mL) for 6 h. (B) Quantification of p62⁺ dots in BMDMs stimulated as in A. (C) Flow cytometry analysis of ROS production in BMDMs pretreated or not with NAC and stimulated with heme (100 μM). (D) Immunofluorescence images of p62⁺ dot (red) formation in BMDMs pretreated with NAC (10 mM) or mitoTEMPO (500 μM) for 1 h and stimulated with heme (100 μM) for 6 h. (E) Quantification of p62⁺ dots in BMDMs stimulated as in D. (F–H) Fluorimetric analysis of ROS production in BMDMs stimulated with heme (100 μM) (F), menadione (100 μM) (G), or H₂O₂ (400 μM) (H). (I–K) Quantification of p62⁺ dots in BMDMs stimulated as in F, G, and H. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. 2.

ROS and NRF2 control heme-induced ALIS formation. (A) Immunofluorescence microscopy showing p62 (red) and Ub (green) localization in BMDMs pretreated or not with NAC (10 mM) for 1 h and stimulated with heme (100 μM) for 6 h. (B and C) Quantification of p62⁺ (A) and Ub⁺ (B) BMDMs stimulated as in A. (D) Confocal microscopy images of BMDMs stained for NRF2 (red) and Ub (green) after stimulation with CoPP (100 μM) for 6 h or with heme (100 μM) for 2, 4, and 6 h. (E and F) Western blot analysis of p62 (E) and Nqo1 (F) protein levels in cell extracts of BMDMs stimulated with CoPP (100 μM) for 6 h or with heme (100 μM) for 2, 4, and 6 h. Actin served as a loading control. (G) Western blot analysis of p62 expression levels in BMDMs from WT and Nrf2^−/− mice stimulated with heme (100 μM) for 2, 4, and 6 h. Actin served as a loading control. (H and I) Immunofluorescence microscopy of BMDMs from WT (H) and Nrf2^−/− (I) mice stimulated with heme (100 μM) or with CoPP (100 μM) for 6 h. (J) Quantification of p62⁺ dots in BMDMs from WT and Nrf2^−/− mice stimulated with heme (100 μM) for 2, 4, and 6 h and with CoPP (100 μM) for 6 h. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. S4.

Heme-induced ALIS requires synthesis of p62. (A) Western blot analysis of p62 protein expression in BMDMs stimulated with heme (30 and 100 μM) for 6 h. Actin served as a loading control. (B) Western blot analysis of p62 protein levels from extracts of BMDMs pretreated with NAC and stimulated with heme (100 μM) for 6 h. Actin served as a loading control. (C) Immunofluorescence microscopy for p62 (red) and Ub (green) of BMDMs pretreated with actinomycin D (ACT D; 5 μg/mL) or cyclohexamide (CHX; 5 μg/mL) and stimulated with heme (100 μM) for 6 h. (D) Quantification of p62⁺ dots of BMDMs stimulated as in C. (E) Western blot analysis of p62 protein levels in extracts from BMDMs stimulated as in A. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. S5.

HO-1 enzymatic activity and heme-derived iron are necessary for ALIS formation. (A and B) Western blot analysis from extracts of BMDMs stimulated by heme (100 μM) for 2, 4, and 6 h (A) or by heme (30, 50, and 100 μM) for 6 h (B). Actin served as a loading control. (C and D) Quantification of p62⁺ (C; red) and Ub⁺ (D; green) dots in BMDMs pretreated or not with SnPP (100 μM) and stimulated with heme (100 μM) for 6 h. (E) Quantification of p62⁺ dots in BMDMs stimulated with heme (100 μM) in the presence or absence of DFO (2 mM 1 h before). (F) Immunofluorescence images showing the colocalization of p62⁺ (red) and Ub⁺ (green) dots in BMDMs stimulated as in E. (G) Schematic representation of heme (Top) and PPIX (Bottom). Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. 3.

HO-1 and iron released from heme are essential for ALIS formation induced by heme. (A) Western blot analysis of HO-1 and p62 expression in cell extracts from Hmox1^+/+, Hmox1^+/−, and Hmox1^−/− BMDMs stimulated with heme (100 μM) for 4 and 6 h. Actin served as a loading control. (B) Quantification of p62⁺ dots in from Hmox1^+/+, Hmox1^+/−, and Hmox1^−/− BMDMs stimulated as in A. (C and D) Immunofluorescence microscopy for p62 (red) and Ub (green) in Hmox1^+/+ (C) and Hmox1^−/− (D) BMDMs. (E) Immunofluorescence microscopy images of BMDMs stained for p62 (red) and Ub (green) after stimulation with heme (100 μM), heme plus deferoxamine (H+DFO), or PPIX (100 μM) for 6 h. (F) Quantification of p62⁺ dots in BMDMs stimulated as described in E. (G and H) Western blot analysis of HO-1 (G) and p62 (H) of cell extracts from BMDMs stimulated with heme (100 μM), H+DFO, PPIX (100 μM), FeCl₃ (Fe³⁺; 100 μM), or FeSO₄ (Fe²⁺; 100 μM) for 6 h. Actin served as a loading control. (I) Immunofluorescence images of the colocalization of (red) and Ub (green) in BMDMs stimulated with FeCl₃ (Fe³⁺; 100 μM) or FeSO₄ (Fe²⁺; 100 μM) for 6 h. (J) Quantification of p62⁺ dots in BMDMs stimulated as in I. (K) Quantification of p62⁺ dots in WT and Nrf2^−/− BMDMs stimulated with FeCl₃ (Fe³⁺; 100 μM) or FeSO₄ (Fe²⁺; 100 μM). (L and M) Quantification of p62⁺ dots in BMDMs stimulated with FeCl₃ (Fe³⁺; 100 μM) or FeSO₄ (Fe²⁺; 100 μM) with or without succinylacetone (SA; 1 μM 1 h before) for 6 h (L) and 24 h (M). DAPI was used to stain nuclei (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. S6.

Fe²⁺ and Fe³⁺ induce ALIS. (A and B) Quantification of p62⁺ dots in BMDMs stimulated with Fe²⁺ (A) or Fe³⁺ (B) (25, 50, or 100 μM) for 2, 4, 6, 12, or 24 h. (C) Quantification of GFP-LC3⁺ dots in RAW cells pretreated with vehicle or wortmannin (200 nM) and stimulated with Fe²⁺ (100 μM) for 12 or 24 h. (D and E) Flow cytometry analysis for ROS production in BMDMs stimulated with Fe²⁺ (100 μM) (D) or Fe³⁺ (100 μM) (E). Results are representative of one of at least three independent experiments. For all quantification experiments, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. 4.

Ferritin is essential to avoid iron-induced ALIS. (A) Western blot analysis of cell extracts from BMDMs stimulated with heme (100 μM), H+DFO (2 mM 1 h before), PPIX (100 μM), FeCl₃ (Fe³⁺; 100 μM), or FeSO₄ (Fe²⁺; 100 μM) for 6 h. Actin served as a loading control. (B) Western blot analysis of p62 protein expression in BMDMs from WT and FtH^−/− BMDMs stimulated with Fe²⁺ or Fe³⁺ (100 μM) for 6 h. Actin served as a loading control. (C and D) Immunofluorescence images showing the colocalization of p62 (red) and Ub (green) in WT (C) and FtH^−/− (D) BMDMs stimulated with Fe²⁺ (100 μM) for 6 h. (E) Quantification of p62⁺ dots in an experiment performed as in C and D. Nuclei were stained with DAPI (blue). (Scale bars: 20 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least 300 cells were counted. Data are mean ± SD. *P < 0.05.

Fig. 5.

Hemolysis triggers ALIS formation in vivo. (A) Western blot analysis of p62 expression levels in the kidneys (Top), spleen (Middle), and liver (Bottom) from control (−) and PHZ-injected mice. Actin served as a loading control. (B and C) Immunofluorescence of spleen (B) and liver (C) tissue sections from control and PHZ-injected mice stained for p62 (green). (D and E) Quantification of p62⁺ dots per field of view (FOV) in spleen and liver tissue sections of control (−) and PHZ-injected mice. (Scale bars: 10 μm.) Results are representative of one of at least three independent experiments. For all quantification experiments using microscopy, at least five fields were counted. Data are mean ± SD. *P < 0.05.