Fiorillo, Stefania (2016) STUDY OF THE MECHANISMS REGULATING Na+/Ca2+ EXCHANGER 3 LOCALIZATION IN MITOCHONDRIA AND ITS PATHOPHYSIOLOGICAL IMPLICATIONS. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: STUDY OF THE MECHANISMS REGULATING Na+/Ca2+ EXCHANGER 3 LOCALIZATION IN MITOCHONDRIA AND ITS PATHOPHYSIOLOGICAL IMPLICATIONS
Creators:
CreatorsEmail
Fiorillo, Stefaniastefania.fiorillo@unina.it
Date: 31 March 2016
Number of Pages: 72
Institution: Università degli Studi di Napoli Federico II
Department: Neuroscienze e Scienze Riproduttive ed Odontostomatologiche
Scuola di dottorato: Medicina molecolare
Dottorato: Neuroscienze
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
nomeemail
Annunziato, Luciolannunzi@unina.it
Tutor:
nomeemail
Molinaro, PasqualeUNSPECIFIED
Date: 31 March 2016
Number of Pages: 72
Uncontrolled Keywords: Mitochondria, NCX3
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Date Deposited: 11 Apr 2016 12:40
Last Modified: 21 Apr 2017 01:00
URI: http://www.fedoa.unina.it/id/eprint/10965

Abstract

Several proteins are localized in two cellular compartments at the same time with special targeting signals that are localized at the N-terminal domain. Among these protein, the isoform 3 of the Na+/Ca2+ exchanger (NCX3) family has been found on both plasma membrane and outer mitochondrial membrane (OMM), where it plays an important role in the maintenance of cytosolic and mitochondrial Ca2+ homeostasis. In particular, mitochondrial NCX3 takes part in mitochondrial Ca2+ efflux from the OMM, promoting cell survival during hypoxia, through an AKAP121-anchored signaling complex. Interestingly, the amount of NCX3 localized on the OMM of neurons decreases during oxygen glucose deprivation and is restored after re-oxygenation (OGD-Rx), suggesting the presence of a regulatory mechanism the subcellular sorting of NCX3. The aim of the present study was to investigate the molecular determinants and the mechanism regulating the subcellular sorting of NCX3 on mitochondria. Results obtained showed that the silencing of either translocase of outer mitochondrial membrane (TOM), TOM22 or TOM70, decreased the amount of NCX3 on mitochondria suggesting that these two mitochondrial import receptors are involved in the recognition and transport of this antiporter on the OMM. By contrast, the silencing of the other mitochondrial import receptor, TOM20, did not show affect the subcellular localization of the antiporter. By using a bioinformatics approach, we identified some putative regions of NCX3 that might contain a mitochondrial localization signal. On the basis of these data, we analyzed the mitochondrial fraction of baby hamster kidney (BHK) cells stably transfected with several chimeric proteins containing the sequences of NCX3 and NCX1 and NCX3 mutants. Mitochondrial extraction of these chimeric proteins revealed that NCX3 is provided with three independent regions containing the molecular determinants for its subcellular localization on OMM and that are localized in the C-terminal region of the cytosolic f-loop (718-756 aa), in the alpha1 (109-133 aa) and alpha2 (788-829 aa) sequences. Furthermore, we generated several site-directed mutants of NCX1 in which single amino acids in the alpha1 or alpha2 region was substituted with the corresponding one present in NCX3. Results showed that, chimeric proteins provided with the backbone of NCX1 and amino acids regions 109-133, 718-787, 788-829 of NCX3 were localized on mitochondria, supporting the conclusion that there are multiple mitochondrial localization signals spread along the protein. Another aspect that emerged from these results is the proximity of the mitochondrial localization signal and the N-terminal signal peptide for plasma membrane in NCX3 sequence as it occurs in other proteins with multiple subcellular localizations. In fact, post-translational modifications, i.e. PKA phosphorylation or cleavage of the N-terminus close to the mitochondrial localization sequence, can regulate the protein topology inside the cell. In this regards, we excluded the participation of PKA phosphorylation in NCX3 topology on mitochondria, since the specific inhibitor of PKA phosphorylation, KT5720, did not influence the distribution of the antiporter between the plasma membrane and mitochondria. On the other hand, the removal of the signal peptide at the N-terminus (NCX3Δ2-31), increased the amount of the exchanger on mitochondria, supporting the hypothesis that the cleavage of this signal peptide, occurring during the antiporter synthesis, can participate in the regulation of NCX3 topology under physiological or pathophysiological conditions.

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