Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80kDa catalytic subunit to a 75kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of this study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80kDa proenzyme in the absence of Ca2+. Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75kDa form. Taken together our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physio-pathological conditions having altered Ca2+ homeostasis.

Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

Roberta De Tullio;Alice Franchi;Antonino Martines;Monica Averna;Marco Pedrazzi;Edon Melloni;Bianca Sparatore
2018-01-01

Abstract

Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80kDa catalytic subunit to a 75kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of this study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80kDa proenzyme in the absence of Ca2+. Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75kDa form. Taken together our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physio-pathological conditions having altered Ca2+ homeostasis.
File in questo prodotto:
File Dimensione Formato  
2018 De Tullio, R.ab_Unexpected-role-of-the-Ldomain-of-calpastatin-during-the-autoproteolytic-activation-of-human-erythrocyte-calpainArticle_2018.pdf

accesso aperto

Descrizione: articolo pubblicato in versione .PDF
Tipologia: Documento in versione editoriale
Dimensione 1.05 MB
Formato Adobe PDF
1.05 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/900994
Citazioni
  • ???jsp.display-item.citation.pmc??? 0
  • Scopus 3
  • ???jsp.display-item.citation.isi??? 3
social impact