CANX Knockout Cell Line (HEK293)

Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully understood. Here, we establish a cell-free system to mimic the activation of MTORC1, by which we identify CANX (calnexin) as an essential regulator for leucine-stimulated MTORC1 pathway. CANX translocates to lysosomes after leucine deprivation, and its loss of function renders either the MTORC1 activity or the lysosomal translocation of MTOR insensitive to leucine deprivation. We further find that CANX binds to LAMP2 (lysosomal associated membrane protein 2), and LAMP2 is required for leucine deprivation-induced CANX interaction with the Ragulator to inhibit Ragulator activity toward RRAG GTPases. Moreover, leucine deprivation promotes the lysine (K) 525 crotonylation of CANX, which is another essential condition for the lysosomal translocation of CANX. Finally, we find that KAT7 (lysine acetyltransferase 7) mediates the K525 crotonylation of CANX. Loss of KAT7 renders the MTORC1 insensitivity to leucine deprivation. Our findings provide new insights for the regulatory mechanism of the leucine-stimulated MTORC1 pathway. CALR: calreticulin; CANX: calnexin; CLF: crude lysosome fraction; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; GST: glutathione S-transferase; HA: hemagglutinin; HEK293T: human embryonic kidney-293T; KAT7: lysine acetyltransferase 7; Kcr; lysine crotonylation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LAMTOR/Ragulator: late endosomal/lysosomal adaptor: MAPK and MTOR activator; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PTM: post-translational modification; RPS6KB1/p70S6 kinase 1: ribosomal protein S6 kinase B1; RPTOR: regulatory associated protein of MTOR complex 1; SESN2: sestrin 2; TMEM192: transmembrane protein 192; ULK1: unc-51 like autophagy activating kinase 1.

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP, CANX, and CALR cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the -Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.