New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET Myriam Salameh, Sylvie Riquier, Olivier Guittet, Meng-Er Huang, Laurence Vernis, Michel Lepoivre, Marie-Pierre Golinelli-Cohen To cite this version: Myriam Salameh, Sylvie Riquier, Olivier Guittet, Meng-Er Huang, Laurence Vernis, et al.. New In- sights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Ho- molog mitoNEET. Biomedicines, MDPI, 2021, 9 (4), 10.3390/biomedicines9040384. hal-03312162 HAL Id: hal-03312162 https://hal.archives-ouvertes.fr/hal-03312162 Submitted on 2 Aug 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. biomedicines Article New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET Myriam Salameh, Sylvie Riquier, Olivier Guittet, Meng-Er Huang, Laurence Vernis, Michel Lepoivre and Marie-Pierre Golinelli-Cohen * CNRS, Institut de Chimie des Substances Naturelles, Université Paris-Saclay, UPR 2301, 91198 Gif-sur-Yvette, France; [email protected] (M.S.); [email protected] (S.R.); [email protected] (O.G.); [email protected] (M.-E.H.); [email protected] (L.V.); [email protected] (M.L.) * Correspondence: [email protected]; Tel.: +33-1-6982-3012 Abstract: Human CISD2 and mitoNEET are two NEET proteins anchored in the endoplasmic reticulum and mitochondria membranes respectively, with an Fe–S containing domain stretching out in the cytosol. Their cytosolic domains are close in sequence and structure. In the present study, combining cellular and biochemical approaches, we compared both proteins in order to possibly identify specific roles and mechanisms of action in the cell. We show that both proteins exhibit a high intrinsic stability and a sensitivity of their cluster to oxygen. In contrast, they differ in according to expression profiles in tissues and intracellular half-life. The stability of their Fe–S cluster and its ability to be transferred in vitro are affected differently by pH variations in a physiological and Citation: Salameh, M.; Riquier, S.; pathological range for cytosolic pH. Finally, we question a possible role for CISD2 in cellular Fe–S Guittet, O.; Huang, M.-E.; Vernis, L.; cluster trafficking. In conclusion, our work highlights unexpected major differences in the cellular Lepoivre, M.; Golinelli-Cohen, M.-P. and biochemical features between these two structurally close NEET proteins. New Insights of the NEET Protein CISD2 Reveals Distinct Features Keywords: iron-sulfur protein; CISD2; Fe–S cluster transfer; Fe–S cluster lability; Wolfram syndrome; Compared to Its Close Mitochondrial UV-visible absorption spectroscopy Homolog mitoNEET. Biomedicines 2021, 9, 384. https://doi.org/ 10.3390/biomedicines9040384 1. Introduction Academic Editor: Stefano Bellosta Recently discovered NEET proteins are iron–sulfur (Fe–S) cluster-containing proteins that are present in all the kingdoms of life. Members of this family are characterized by Received: 12 February 2021 at least one highly conserved 39-amino-acid motif called CDGSH Iron–Sulfur Domain Accepted: 3 April 2021 Published: 5 April 2021 (CISD) harboring a redox-active 2Fe–2S cluster coordinated by an unusual 3Cys–1His quartet instead of the usual 4Cys. In mammals, three NEET proteins have been identified: Publisher’s Note: MDPI stays neutral mitoNEET (also known as CISD1), CISD2 (NAF-1, ERIS, Noxp70 or Miner1) and CISD3 with regard to jurisdictional claims in (Miner2 or MiNT) [1,2]. Even if their specific functions are still debated, they are clearly published maps and institutional affil- involved in various processes including ageing, autophagy, apoptosis and the regulation of iations. calcium, iron metabolism, Fe–S cluster and reactive oxygen species (ROS) homeostasis. The molecular mechanisms underlying the involvement of NEET proteins in these multiple processes remain poorly understood [2]. Several studies reveal that various types of cancers are accompanied by overexpression of NEET proteins [3] and the CISD2 expression level has been proposed as an independent Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. prognostic marker for survival in cancer patients including pancreatic [4] and gastric can- This article is an open access article cers [5]. More generally, the overexpression of NEET proteins promotes the proliferation distributed under the terms and of cancer cells, supports tumor growth and metastasis. Conversely, their depletion leads conditions of the Creative Commons to decreased cancer cell proliferation in breast cancer [6] and neuroblastomas [7]. Reces- Attribution (CC BY) license (https:// sive mutations in the CISD2 gene coding for CISD2 are the causative factor for Wolfram creativecommons.org/licenses/by/ syndrome 2 (or WFS2), a rare autosomal recessive neurodegenerative disorder leading to 4.0/). severe neurological disabilities and a drastic decrease in life span [8]. Biomedicines 2021, 9, 384. https://doi.org/10.3390/biomedicines9040384 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 16 Biomedicines 2021, 9, 384 2 of 16 Wolfram syndrome 2 (or WFS2), a rare autosomal recessive neurodegenerative disorder leading to severe neurological disabilities and a drastic decrease in life span [8]. MitoNEETMitoNEET and and CISD2 CISD2 are are dimers dimers that that assemble assemble one one cluster cluster per per protomer protomer whereas whereas CISD3CISD3 is monomericis monomeric and and assembles assembles two two clusters. clusters. While the the latter latter resides resides inside inside the the mito- mitochondrialchondrial matrix, matrix, mitoNEET mitoNEET and and CISD2 CISD2 are are re respectivelyspectively bound bound to to the the mitochondrial mitochondrial and andendoplasmic endoplasmic reticulum reticulum (ER) (ER) membranes membranes by bya sp aecific specific N-terminal N-terminal anchor anchor with with the remain- the remaininging parts parts of ofthe the protein, protein, including including the the Fe–S clustercluster domain, domain, laying laying into into the the cytosol cytosol [2]. [2]. TheirTheir cytosolic cytosolic domains domains share share 65% 65% identity identity and an 79%d 79% similarity similarity (Figure (Figure1) with 1) with a similar a similar uniqueunique folding folding that that exhibits exhibits two two distinct distinct domains: domains: the Fe–Sthe Fe–S cluster cluster binding binding domain domain and and a a β-capβ-cap domain domain [2]. [2]. Figure 1. Comparison of the amino acid sequences of human mitoNEET (NP_060934.1, 12.2 kDa) and CISD2 (NP_001008389.1, 15.3 kDa). The following markup lines are indicated: “.”, small positive score; “:”, similarity; “|”, identity. Non-cytosolic parts of the proteins (in-organelle domain and membrane anchors) are marked by grey boxes. Figure 1. Comparison of the amino acid sequences of human mitoNEET (NP_060934.1, 12.2 kDa) and CISD2 Residues involved in cluster coordination are in bold. The C92S point mutation introduced to express the cytosolic domain (NP_001008389.1, 15.3 kDa). The following markup lines are indicated: “.”, small positive score; “:”, similarity; “|”, iden- of CISD2 is indicated by an arrow. tity. Non-cytosolic parts of the proteins (in-organelle domain and membrane anchors) are marked by grey boxes. Residues involved in cluster coordination are in bold. The C92S point mutation introduced to express the cytosolic domain of CISD2 One of the most outstanding biochemical properties of the NEET proteins is their is indicated by an arrow. ability to transfer their cluster to acceptor apoproteins (cluster transfer reaction) [9,10]. By combining in vitro and in cellulo approaches, our recent data demonstrated the involve- One of the most outstanding biochemical properties of the NEET proteins is their ment of the human mitoNEET in a novel Fe–S trafficking pathway to quickly rebuild a ability to transfer their cluster to acceptor apoproteins (cluster transfer reaction) [9,10]. By cluster in IRP-1/cytosolic aconitase following an oxidative insult [11]. Remarkably, the combining in vitro and in cellulo approaches, our recent data demonstrated the involve- mitoNEET cluster is extremely stable when reduced, even at acidic pH, and can hardly be ment of the human mitoNEET in a novel Fe–S trafficking pathway to quickly rebuild a lost or transferred [12]. However, it can be reversibly oxidized to [2Fe–2S]2+ by hydrogen cluster in IRP-1/cytosolic aconitase following an oxidative insult [11]. Remarkably, the mi- peroxide (H O ) and be further reduced by biological thiols suggesting a redox sensory toNEET 2cluster2 is extremely stable when reduced, even at acidic pH, and can hardly be function of mitoNEET [13,14]. Only an oxidized mitoNEET cluster can be transferred lost or transferred [12]. However, it can be reversibly oxidized to [2Fe–2S]2+ by hydrogen in vitro to physiological cluster acceptors such as the cytosolic aconitase and to