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Heme-Iron Biology Unit

Emanuela Tolosano - PI

Emanuela Tolosano
Biographical sketch

Full Professor Biology, University of Torino, School of Medicine, Italy

Main group members
Main group members
  • Veronica Fiorito, Senior Post-doc
  • Sara Petrillo, Senior Post-doc
  • Francesco De Giorgio, Dottorando
  • Carola Ronco, Dottoranda

Research activity

Heme, an iron-containing porphyrin, serves as the prosthetic group of proteins involved in various biological processes, such as oxygen transport and storage, cell respiration, drug metabolism, and gene expression control. Its role in regulating oxidative metabolism is crucial, as heme influences oxygen availability, electron transport chain complexes activity, and participates in tricarboxylic acid (TCA) cycle cataplerosis. Consequently, it is not surprising that disruptions in heme metabolism occur in numerous pathological conditions, including genetic diseases, hematological disorders, and cancer.

Maintaining cellular heme homeostasis relies on the coordinated expression and activity of enzymes and transporters involved in heme synthesis, acquisition from external sources, degradation, and transport within subcellular compartments and across the plasma membrane (Figure 1. Cellular heme homeostasis).

 

Heme is biosynthesized through a series of reactions occurring partly in mitochondria and partly in the cytosol (1). The first rate-limiting step of heme biosynthesis, the condensation of glycine with the tricarboxylic acid cycle-derived Succinyl-CoA, is catalyzed by the enzyme ALAS1. The intermediates are then transformed by a series of reactions occurring partly in mitochondria and partly in the cytosol, catalyzed by ALAD, PBGD, UROS, UROD, CPOX and PPOX. The last reaction, catalyzed by FECH, consists in the insertion of iron (Fe2+) in the protoporphyrin ring. Newly synthesized heme can be transferred to endoplasmic reticulum (ER) for incorporation into hemoproteins (2), to the nucleus (3) where it can regulate specific transcription factors like BACH1, Rev-Erbα and p53, or to the cytosol. Various membrane transporters, such as HRGs, ABCG2, and ABCC5, facilitate the transport of heme or its precursors across cellular membranes. Additionally, transporters like FLVCR1 and FLVCR2 contribute to the regulation of heme homeostasis, even though they do not directly transport heme or its precursors (4, 5). Additionally, heme can be obtained through the endocytosis of senescent red blood cells or receptor-mediated endocytosis of hemoglobin-haptoglobin and heme-hemopexin complexes (not shown). Intracellular heme can be imported into the cytosol via HRG1 on endolysosomal membranes and degraded by HMOX-1 in the ER (6).

ALAS1 = 5-aminolevulinate synthase 1, ALAD = aminolevulinate dehydratase, PBGD = porphobilinogen deaminase, UROS = uroporphyrinogen III Synthase, UROD = uroporphyrinogen III decarboxylase, CPOX = coproporphyrinogen oxidase, PPOX = protoporphyrinogen oxidase, FECH = ferrochelatase, BACH1 = BTB Domain And CNC Homolog 1, Rev-Erbα = nuclear receptor subfamily 1 group D member 1, p53 = transformation-related protein 53, FLVCR1 = Heme Transporter 1, FLVCR2 = FLVCR Heme Transporter 2, ABCG2 = ATP binding cassette subfamily G member 2, , HRG1 = solute carrier family 48 member 1,  MRP5 = ABCC5: ATP binding cassette subfamily C member 5, HMOX1 = heme oxygenase 1. (Created by Biorender).

Our long-term research objective is to elucidate the mechanisms that regulate cellular heme homeostasis. By comprehending how heme homeostasis is upheld in healthy individuals and disrupted in pathological conditions, we aim to develop innovative therapeutic approaches.

Our research is primarily focused on characterizing the functional interplay between 5-aminolevulinate synthase 1 (ALAS1), the initial and rate-limiting enzyme in the heme biosynthetic pathway, and Feline Leukemia Virus C Receptor 1 (FLVCR1), a plasma membrane importer of choline and ethanolamine that positively regulates ALAS1 activity. Through gene silencing techniques, genetic manipulation, and pharmacological interventions, we have established various cellular and animal models that either inhibit or enhance ALAS1-FLVCR1 system (Figure 2  Cellular and animal models available in the lab). 

We have developed cancer cell lines with modified heme synthesis-export systems, enabling us to study both inhibitory and enhancing effects. Additionally, we have created mouse models with knockout, knockin, or conditional alleles that perturb the heme system. Furthermore, we have zebrafish models with impaired heme synthesis. These models provide valuable tools for studying the underlying mechanisms. Cancer cell lines can be utilized to generate xenografts in mice, while primary cell cultures derived from the mouse models allow for detailed investigations into the molecular aspects of heme regulation. (Created by Biorender).

These models have allowed us - and will continue to help us - to: (i) identify biological processes that depend on a functional ALA1-FLVCR1 system (as exemplified in Figure 3), (ii) define the mechanisms underlying these processes, and (iii) design strategies to restore heme homeostasis and regain a healthy state or (iv) exploit heme dependencies to selectively target pathological cells such as cancer cells.

Figure 3

Figure 3. Defective angiogenesis in endothelial specific Flvcr1a-null mice. 

The retinas were isolated from endothelial specific Flvcr1a-null pups and controls at P8 and stained with an antibody against the endothelial marker CD31. Scale bar: 100 µm. Deletion of FLVCR1a in endothelial cells results in impaired heme metabolism that cannot sustain the proper development of the vascular network.
 

Our objective is to unravel the connection between choline/ethanolamine import and heme synthesis, focusing on how the ALAS1-FLVCR1 system governs cellular energy metabolism and shapes biological outcomes. In particular, we aim to: 

(i) assess the influence of FLVCR1-regulated heme metabolism on the advancement and progression of tumors and leverage this system as a potential target for therapeutic interventions; 

(ii) gain a deeper understanding of the dependency of endothelial cells on a functional ALAS1-FLVCR1 system to support both physiological and pathological angiogenesis; 

(iii) evaluate the potential impact of ALAS1-FLVCR1 system on liver and skeletal muscle, leading to alterations in glucolipid metabolism and potentially influencing susceptibility to the development of metabolic syndrome.

  • Title of contract: Exploiting heme-driven metabolic rewiring in tumor cell and tumor endothelial cell to control lung cancer progression. 2021-2026 From: AIRC Financing: 468000 Euro Principal Investigator: Emanuela Tolosano
  • Title: Defective heme transport in the development of congenital hydrocephalus.  2021-2026 From: NIH, 1R01NS123168-01.  Financing: 300000 USD Role: Co-PI
  • Title: 2022XJNWRM: Disentangling genetic, epigenetic and hormonal regulation of Fe/heme metabolism in the gender-specific nature of NAFLD (DEFENDER). 2023-2025 From: MUR Italian Ministry of University and Research Financing: 94000 Euro Role: PI
  • Title: P2022KHR59: Unsolved challenges in metabolic syndrome: the role of heme metabolism in liver and muscle gluco-lipid homeostasis. 2023-2025 From: MUR Italian Ministry of University and Research Financing: 101900 Euro Role: PI
  1. Bertino F., Mukherjee D., Bonora M., Bagowski C., Nardelli J., Metani L., Zanin Venturini D.I., Chianese D., Santander N., Salaroglio I.C.,  Hentschel A., Quarta E., Genova T., McKinney A.A., Allocco A.L., Fiorito V., Petrillo S., Ammirata G., De Giorgio F., Dennis E., Allington G., Maier F., Shoukier M., Gloning K-P., Munaron L, Mussano F., Salsano E., Pareyson D., Di Rocco M., Altruda F., Panagiotakos G., Kahle K.T., Gressens P., Riganti C., Pinton P.P., Roos A., Arnold T., Tolosano E., Chiabrando D.,” Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus”, Cell Reports Medicine 5, 101647 (2024). https://doi.org/10.1016/j.xcrm.2024.101647
  2. Mistretta M., Fiorito V., Allocco A.L., Ammirata G., Hsu M.Y., Digiovanni S., Belicchi M., Napoli L., Ripolone M., Trombetta E., Mauri P., Farini A, Meregalli M., Villa C., Porporato P.E., Miniscalco B., Geninatti Crich S., Riganti C., Torrente Y., Tolosano E., “Flvcr1a deficiency promotes heme-based energy metabolism dysfunction in skeletal muscle”, Cell Reports 43(3):113854 (2024). https://doi.org/10.1016/j.celrep.2024.113854 
  3. Petrillo S., De Giorgio F., Bertino F., Garello F., Bitonto V., Longo D.L., Mercurio S., Ammirata  G., Allocco A.L., Fiorito V., Chiabrando D., Altruda F., Terreno E., Provero P., Munaron L., Genova T., Nóvoa A., Carlos A.R., Cardoso S., Mallo M., Soares M.P., Tolosano E., “Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis”, Angiogenesis doi: 10.1007/s10456-023-09865-w (2023) https://doi.org/10.1007/s10456-023-09865-w
  4. Allocco, A.L., Bertino, F., Petrillo, S., Chiabrando, D., Riganti, C., Bardelli, A., Altruda, F., Fiorito, V., Tolosano, E., “Inhibition of Heme Export and/or Heme Synthesis Potentiates Metformin Anti-Proliferative Effect on Cancer Cell Lines”. Cancers 2022, 14, 1230 (2022). https://doi.org/10.3390/cancers14051230
  5. Fiorito V., Allocco A.L., Petrillo S., Gazzano E., Torretta S., Marchi S., Destefanis F., Pacelli C., Audrito V., Provero P., Medico E., Chiabrando D., Porporato P.E., Cancelliere C., Bardelli A., Trusolino L., Capitanio N., Deaglio S., Altruda F., Pinton P., Cardaci S., Riganti C., Tolosano E., “The heme synthesis-export system regulates the tricarboxylic acid cycle flux and oxidative phosphorylation”, Cell Reports 35(11):109252. https://doi.org/10.1016/j.celrep.2021.109252
  6. Petrillo S., Chiabrando D., Genova T., Fiorito V., Ingoglia G., Vinchi F., Mussano F., Carossa S., Silengo L., Altruda F., Merlo G.R., Munaron L., Tolosano E., “Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis”, Cell Death & Differentiation 25:573–588 (2018). https://doi.org/10.1038/s41418-017-0001-7
  7. Chiabrando D., Castori M., Di Rocco M., Voigt M., Gießelmann S., Di Capua M., Madeo A., Grammatico P.,Hübner C.A., Altruda A., Silengo L., Tolosano E.+, Kurth I.+, “MUTATIONS IN THE HEME EXPORTER FLVCR1 CAUSE SENSORY NEURODEGENERATION WITH LOSS OF PAIN PERCEPTION', PLOS Genetics 12(12):e1006461 (2016). https://doi.org/10.1371/journal.pgen.1006461
  8. Vinchi F., Costa da Silva M., Ingoglia G., Petrillo S., Brinkman N., Zuercher A., Cerwenka A., Tolosano E.+, Muckenthaler M.U.+, “Hemopexin therapy reverts heme-induced pro-inflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease”, Blood 127(4): 473-486 (2016) https://doi.org/10.1182/blood-2015-08-663245
  9. Fiorito V., Forni M., Silengo L., Altruda F., Tolosano E., “Crucial role of Flvcr1a in the maintenance of intestinal heme homeostasis”, Antioxidants & Redox Signaling 23(18): 1410-1423 (2015). https://doi.org/10.1089/ars.2014.6216
  10. Vinchi F., Ingoglia G., Chiabrando D., Mercurio S., Turco E., Silengo L., Altruda F., Tolosano E., “Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450”, Gastroenterology 146: 1325–1338 (2014). https://doi.org/10.1053/j.gastro.2014.01.053
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