MECHANISM OF CALCIUM RELEASE DURING UNFOLDED PROTEIN RESPOSE

FELIZIANI, COSTANZA; QUASSOLLO, GONZALO; BOLLO, MARIANA

Congreso; Sociedad Argentina de Investigación en Neurociencias (SAN) XXXIII Annual Meeting.; 2018

Sociedad Argentina de Investigación en Neurociencias

The Endoplasmic Reticulum (ER) is a multi-functional organelle that plays a critical role in a variety of processes, where the ER Ca 2+ acts as a key messenger. Under resting conditions, the luminal Ca 2+ concentration reflectsa balance between active uptake by Ca 2+ -ATPases and passive efflux pathways of which the translocon can play a prominent role. The translocon is an aqueous pore, primarily formed by the Sec61 core spanning the ERlipid bilayer, that is blocked by the ribosome on the cytosolic side and by the ER chaperone, BiP, on the luminal side. We hypothesize that during the acute phase of the UPR (Unfolded Protein Response), immediately afteraccumulation of unfolded protein in the lumen, the Ca 2+ ER eflux through the translocon is increased. To test this mechanism, we performed cytosolic Ca 2+ measurements in primary cultures of human astrocytes,expressing the Ca 2+ indicator GCaMP6 tethered to the ER membrane, after induction of the UPR by Tunicamycin (Tm, glycosylation inhibitor). We observed focal release of Ca 2+ (microdomains) in stressed astrocytes thatwas significantly inhibited by translocon blockers (emetine or anisomycin). In addition, the Tm-induced Ca 2+ signal was amplified by pre-treatment either with AB5 cytotoxine, which specifically hydrolyses BiP, or with thetranslocon opener puromycin. The effect of these pharmacological tools was corroborated by co-immunoprecipitations that showed changes in the interactions either between Sec61α and BiP or Sec61α and the ribosomalprotein S6. Important to note that the likehood of obtained Tm-induce local Ca 2+ events, increase by using either the slow chelator EGTA-AM or Xestospongin C and Ryanodine (InsP 3 and Ry Receptors inhibitors,respectively). Moreover, we specifically plug the pore using a truncated prolactin with 64 amino acid residues lacking the stop codon (Prl-64), thus the nascent chain remains bound to the ribosome as a peptidyl-tRNAblocking the pore. The Prl-64 expression clearly exhibited Tm-induce Ca 2+ local increase. Finally, InsP 3 Receptor null HEK-293 cell line (HEK3KO) exhibited Tm-evoked Ca 2+ release near ER microdomain, which is inhibited byBiP over-expression. Taken together, these data, strongly suggest that the chaperone BiP and the ribosome are dissociated from the translocon increasing Ca 2+ permeability.