Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

Summary TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.

Proprotein convertases (PCs), furin and proprotein convertase 1/3 (PC1), cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts which facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PROFUR), which regulates furin activation at pH~6.5 within the trans Golgi network. Although this residue is conserved in the PC1 propeptide (PROPC1), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here we analyze the mechanism by which PROFUR regulates furin activation and examine why PROFUR and PROPC1 differ in their pH-dependent activation. Sequence analyses establish that while both PROFUR and PROPC1 are enriched in histidines when compared with cognate catalytic-domains and prokaryotic orthologs, histidine content in PROFUR is ~two-fold greater than PROPC1, which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine-protonation significantly unfolds PROFUR when compared to PROPC1 to enhance autoproteolysis. We further demonstrate that PROFUR and PROPC1 are sufficient to confer organelle-sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH-gradients to activate within specific organelles.

Background: Histidine 69 in the propeptide is a pH sensor that mediates compartment-specific furin activation. Results: Histidine 69 protonation exposes the activation loop for proteolysis only within an optimal window for pH-dependent activation. Conclusion: A small structural change functions as the trigger that regulates precise spatiotemporal activation of furin. Significance: Our work provides insights into how individual proprotein convertases encode their unique compartmentspecific activation.