Abstract
ABSTRACTCAPNS1 is essential for stability and function of the ubiquitous calcium-dependent proteases micro- and milli-calpain. Upon inhibition of the endoplasmic reticulum Ca2+ ATPase by 100 nM thapsigargin, both micro-calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1-dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1-containing vesicles with LC3 bodies in control cells. By contrast, CAPNS1 depletion is coupled with an accumulation of LC3 bodies and Rab5 early endosomes. Moreover, Atg9 and Bif-1 remain in the GM130-positive Golgi stacks and Atg9 fails to interact with the endocytic route marker transferrin receptor and with the core autophagic protein Vps34 in CAPNS1-depleted cells. Ectopic expression of a Bif-1 point mutant resistant to calpain processing is coupled to endogenous p62 and LC3-II accumulation. Altogether, these data indicate that calpain allows dynamic flux of Atg9/Bif-1 vesicles from the Golgi toward the budding autophagosome.
Highlights
The ubiquitous μ- and m-calpain are calcium-dependent neutral cysteine proteases composed of an 80 kDa catalytic subunit, CAPN1 or CAPN2, respectively, and a common 28 kDa regulatory subunit, CAPNS1 (Goll et al, 2003)
Thapsigargin triggers calpain activation and autophagy in human U2OS cells We previously showed that CAPNS1 depleted human osteosarcoma U2OS cells and CAPNS1−/− MEFs fail to induce autophagosomes formation in response to nutrient deprivation and rapamycin, the classic autophagy-inducing stimuli. (Demarchi et al, 2006)
Atg9, the only known transmembrane autophagic protein is present both in the endosomal compartment, and Golgi apparatus, both described as autophagosome membrane sources (Orsi et al, 2012; Puri et al, 2013)
Summary
The ubiquitous μ- and m-calpain are calcium-dependent neutral cysteine proteases composed of an 80 kDa catalytic subunit, CAPN1 or CAPN2, respectively, and a common 28 kDa regulatory subunit, CAPNS1 (Goll et al, 2003). Calpains proteolytically process a number of specific substrates, in a tightly regulated manner, and exert pleiotropic functions within the living cell. They modulate the adhesive complex dynamics in adherent cells (Bhatt et al, 2002), exerting both positive and negative functions in cellular adhesion and movement. Calpain can positively regulate autophagy (Demarchi et al, 2006; Yoon et al, 2008; Escalante et al, 2013) and switch it off
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