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Vecellio Reane, Denis (2016) Identification and characterization of a novel MICU1 splice variant. [Tesi di dottorato]

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Abstract (inglese)

The ability of mitochondria to take up Ca2+ plays a fundamental role in the regulation of several biological processes [1]. In the last five years, the molecular and functional characterization of the MCU machinery pictures this Ca2+ channel as one of the most sophisticated ion channels described so far [2]. These groundbreaking discoveries have opened a new era for the study of mitochondrial Ca2+ in cell physiology and will allow to deepen the knowledge on the tissues-specific properties of MCU [3] that are still poorly understood.
In this regard, we identified an alternative splice isoform (hereafter named MICU1.1) of the positive MCU modulator MICU1, characterized by the addition of a micro-exon coding for 4 amino acids (EFWQ), conserved in vertebrates. Interestingly, while MICU1 is ubiquitously expressed, MICU1.1 shows a peculiar tissues distribution, being highly expressed in tissues that display the greatest level of mitochondrial Ca2+ uptake, skeletal muscle and lower levels are found in brain.
Immunoprecipitation experiments performed in HeLa cells assessed that MICU1.1 efficiently interacts with MCU and MICU2. Furthermore, MICU1.1 is able to form homo- and heterodimers with MICU2, as well as MICU1. Nonetheless, the overexpression of MICU1.1 in HeLa cells causes a major increase of mitochondrial Ca2+ uptake upon histamine stimulation compared to conventional MICU1, without affecting neither cytosolic Ca2+ values nor the mitochondrial membrane potential. Strikingly, MICU2 overexpression in cells expressing MICU1.1 is unable to block the increase of mitochondrial Ca2+ uptake induced by MICU1.1. On the contrary, the co-expression of MICU1.1 together with MICU2 further increases mitochondrial Ca2+ uptake speed compared to cells overexpressing MICU1.1 alone. On the other hand, MICU1.1, when bound to MICU2, is able to act as gatekeeper of the channel at resting Ca2+ levels as well as MICU1-MICU2 heterodimer. Importantly, we found that MICU1.1-MICU2 overexpression induces the shift of the threshold of MCU opening towards lower Ca2+ concentrations.
Consistently with previous results on MICU1, MICU1.1 function is dependent on its ability to bind Ca2+. Indeed, a MICU1.1 mutant, insensitive to Ca2+, displays a dominant-negative effect on mitochondrial Ca2+ uptake. On the contrary, MICU1.1 is less affected by the dominant-negative effect of mutated MICU2, insensitive to Ca2+ binding.
We also analysed the contribution of the extra-exon to the particular behaviour of MICU1.1. We observed that single mutations or deletions of these residues do not influence the effect of MICU1.1 on mitochondrial Ca2+ uptake. On the contrary, the substitution of all the four amino acids of the extra-exon with four alanine residues is sufficient to recapitulate MICU1 behaviour.
In conclusion, we characterized a transcript variant of MICU1, which is specifically expressed in excitable tissues, prevalently in skeletal muscle, and that shows a higher ability to activate MCU compared to conventional MICU1. Interestingly, MICU1.1 exerts a peculiar function when bound to MICU2. Overall, our data demonstrate a skeletal muscle-specific mitochondrial Ca2+ uptake machinery with a presumably unique function. Thus, in this tissue, mitochondrial Ca2+ can exert new, unexplored roles. Future experiments have to be performed to clarify its physiological and pathological relevance.

Abstract (italiano)

La capacità dei mitocondri di accumulare Ca2+ riveste un ruolo cruciale nella regolazione di numerosi processi fisiologici [1]. Negli ultimi cinque anni, la caratterizzazione dell’identità molecolare e funzionale del complesso dell’uniporto mitocondriale per il calcio (MCU) ha delineato questo canale come uno dei canali più complessi finora descritti [2]. Questa scoperta rivoluzionaria ha inaugurato una nuova era per lo studio del ruolo del Ca2+ mitocondriale nella fisiologia cellulare e ha permesso di approfondire i meccanismi di regolazione tessuto-specifici di MCU [3], ad oggi ancora poco chiari.
A questo proposito, abbiamo identificato una variante di splicing del modulatore positivo di MCU, MICU1. Questa variante di splicing, che abbiamo chiamato MICU1.1, è il risultato di un evento di splicing alternativo che determina l’aggiunta di un micro-esone, conservato in tutti i vertebrati, codificante per quattro aminoacidi (EFWQ). Mentre MICU1 è espresso, seppur a diversi livelli, in tutti i tessuti, MICU1.1 presenta una distribuzione peculiare. Infatti, MICU1.1 è molto espresso nei tessuti che sono noti avere un elevato ingresso di Ca2+ nei mitocondri, ovvero il muscolo scheletrico e il tessuto nervoso, mentre è assente in tutti gli altri.
Esperimenti di immunoprecipitazione effettuati in cellule HeLa hanno dimostrato che MICU1.1 interagisce con MCU e MICU2. Inoltre, MICU1.1 è in grado di formare omodimeri e eterodimeri con MICU2, come già osservato per MICU1. Nonostante ciò, la sovraespressione di MICU1.1 in cellule HeLa causa un aumento dell’entrata di Ca2+ mitocondriale maggiore rispetto a MICU1, senza influenzare né i valori di Ca2+ citosolici né il potenziale di membrana mitocondriale. Sorprendentemente, la co-espressione di MICU2 in cellule sovraesprimenti MICU1.1 non limita l’incremento di ingresso di Ca2+ nei mitocondri indotto dalla sovraespressione di MICU1.1. Al contrario, la co-espressione di MICU1.1 e MICU2 causa un aumento della velocità di entrata di Ca2+ nei mitocondri, che risulta essere maggiore rispetto a quella osservata in seguito alla sola sovraespressione di MICU1.1. Tuttavia, in condizioni basali, MICU1.1, quando forma eterodimeri con MICU2, causa la chiusura del canale allo stesso modo dell’eterodimero MICU1-MICU2. Ciononostante, abbiamo osservato che la sovraespressione di MICU1.1 con MICU2 induce un abbassamento della soglia di attivazione di MCU verso concentrazioni di Ca2+ più basse.
Come già dimostrato in precedenti studi su MICU1, anche la funzione di MICU1.1 dipende dalla capacità di questa proteina di legare Ca2+. Infatti, un mutante di MICU1.1 insensibile ai livelli di Ca2+, agisce da dominante negativo sull’entrata di Ca2+ nel mitocondrio.
Abbiamo anche dimostrato che il comportamento peculiare di MICU1.1 dipende dai residui che compongono l’esone addizionale. I risultati ottenuti dimostrano che la singola mutazione di uno di questi residui (in particolare la sostituzione con alanina o la delezione) non influenza l’effetto di MICU1.1 sull’ingresso di Ca2+ nel mitocondrio. Tuttavia, la sostituzione di tutti e quattro gli aminoacidi con l’aminoacido alanina è sufficiente a ristabilire il comportamento di MICU1.
In conclusione, durante il mio periodo di dottorato ho caratterizzato una variante di splicing di MICU1, che è selettivamente espressa in tessuti eccitabili e che attiva MCU più efficientemente di MICU1. Sorprendentemente, ho osservato che MICU1.1 esercita una funzione peculiare quando legato a MICU2. Complessivamente, i nostri dati dimostrano che nei tessuti eccitabili esiste un complesso molecolare per l’ingresso di Ca2+ nei mitocondri con una funzione unica. Quindi, in questi tessuti, l’ingresso di Ca2+ nei mitocondri riveste funzioni nuove e ancora inesplorate. Ulteriori studi saranno necessari per chiarire il ruolo di MICU1.1 in diverse condizioni fisiologiche e patologiche.

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Tipo di EPrint:Tesi di dottorato
Relatore:Mammucari, Cristina
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > BIOSCIENZE E BIOTECNOLOGIE > BIOLOGIA CELLULARE
Data di deposito della tesi:29 Gennaio 2016
Anno di Pubblicazione:29 Gennaio 2016
Parole chiave (italiano / inglese):mitchondrial calcium, MCU, MICU1
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/04 Patologia generale
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:9354
Depositato il:06 Ott 2016 14:57
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