The endoplasmic reticulum and the unfolded protein response

Abstract

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.

Figures

Figure 1. Protein Trafficking from the ER

Upon translocation of polypeptides through the Sec61 proteinaceous channel, asparagine residues are frequently modified by covalent addition of a preassembled oligosaccharide core (N-acetylglucosamine2-mannose9-glucose3). This reaction is catalyzed by the oligosaccharyltransferase (OST), a multisubunit complex associated with translocon. To facilitate unidirectional transport through the translocon, nascent polypeptide chains in the ER lumen interact with BiP, a molecular chaperone that binds to exposed hydrophobic residues. Subsequently, rapid deglucosylation of the two outermost glucose residues on the oligosaccharide core structures, mediated by glucosidase I and II (GlcI and GlcII), prepares glycoproteins for association with the ER lectins calnexin and calreticulin. The calnexin/calreticulin-associated oxidoreductase ERp57 facilitates protein folding by catalyzing formation of intra- and inter- molecular disulfide bonds, a rate-limiting step in the protein folding process. Release from calnexin/calreticulin followed by glucosidase II cleavage of the innermost glucose residue prevents further interaction with calnexin and calreticulin. At this point, natively folded polypeptides transit the ER to the Golgi compartment, in a process possibly assisted by mannose-binding lectins, such as ERGIC-53, VIPL, ERGL. As an essential component of protein-folding quality control, non-native polypeptides are tagged for reassociation with calnexin/calreticulin by the UDP-glucose:glycoprotein glucosyltransferase (UGT1) to facilitate their ER retention and prevent anterograde transport. Polypeptides that are folding incompetent are targeted for degradation by retrotranslocation, possibly mediated by EDEM and Derlins, into the cytosol and delivery to the 26S proteosome. Triangles represent glucose residues, squares represent N-acetylglucosamine residues, and circles represent mannose residues.

Figure 2. Signaling the unfolded protein response

Three proximal sensors IRE1, PERK and ATF6 regulate the UPR through their respective signaling cascades. Under non-stressed conditions, BiP binds to the lumenal domains of IRE1 and PERK to prevent their dimerization. Upon accumulation of unfolded proteins in the ER lumen, IRE1 released from BIP dimerizes to activate its kinase and RNase activities to initiate XBP1 mRNA splicing thereby creating a potent transcriptional activator. Primary targets that require IRE1/XBP1 pathway for induction include genes encoding functions in ERAD. Similarly, ATF6 released from BiP transits to the Golgi compartment where for cleavage by S1P and S2P proteases to yield a cytosolic fragment that migrates to the nucleus to further activate transcription of UPR-responsive genes. Finally, PERK released from BiP dimerizes, autophosphorylates, and phosphorylates eIF2α on Ser 51 leading to general attenuation of translational initiation. Paradoxically, eIF2α phosphorylation induces translation of ATF4 mRNA. The PERK/eIF2α/ATF4 regulatory axis also induces expression of anti-oxidative stress response genes and expression of genes encoding proteins with proapoptotic functions, such as CHOP.

Figure 3. Pathways of ER stress-induced apoptosis

ER stress leads to several redundant pathways for caspase activation that involve mitochondrial- dependent and independent pathways. Activated IRE1 recruits TRAF2 to elicit JNK phosphorylation and activation. Caspase 12 is a murine ER-associated proximal effector in the caspase activation cascade that activates procaspase 9 to cleave procaspase 3, the primary executioner of cell death. A second cell death signaling pathway activated by ER stress is mediated by transcriptional induction of genes encoding proapoptotic functions. Activation of PERK, ATF6 and possibly IRE1 lead to transcriptional induction of CHOP that induces apoptosis possibly through up-regulatng expression of the genes Gadd34, Dr5 and Trb3 or by inhibiting expression of the anti-apoptotic gene Bcl2. Mitochondrial ROS can be generated as a result of ER stress-induced Ca+2 release and depolarization of the inner mitochondrial membrane. Thus, oxidative stress in association of unresolved ER stress contributes to multiple pathways of cell death.