The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology

Abstract

Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding "problem," as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.

FIGURE 1. Protein targeting to the endoplasmic reticulum

As a nascent secretory protein emerges from the ribosome exit tunnel, it presents a hydrophobic signal sequence that is recognized by the signal recognition particle (SRP). Binding of SRP to the signal sequence slows translation and targets the ribosome-nascent chain complex to the ER membrane via its interaction with the dimeric SRP receptor. Following release, SRP is recycled and translation resumes. A nascent soluble protein (A) is translocated into the ER lumen, and an integral membrane protein (B) is incorporated into the membrane by the Sec61 translocation complex, and for all soluble proteins and some membrane proteins the hydrophobic signal sequence is cleaved by the ER localized signal peptidase complex.

FIGURE 2. Steps in endoplasmic reticulum-associated degradation (ERAD)

Recognition: during protein synthesis and translocation, a misfolded region (red star) may reside in a protein’s cytoplasmic, ER luminal, or transmembrane domains. Recognition is mediated by ER luminal or cytoplasmic chaperones, as depicted, depending on the location of the folding lesion. For glycoproteins, lectins (pink) interact with N-glycans and in some cases they monitor the folding status of the protein. Ubiquitination: following recognition, the ubiquitination machinery is recruited to the misfolded substrate, either directly within the membrane or by interactions with cytoplasmic chaperones. A ubiquitin activating enzyme (E1) transfers ubiquitin (gray circle) to an active site cysteine in a ubiquitin conjugating enzyme (E2) in an ATP-dependent process. The ubiquitin is then transferred most commonly to a lysine residue on a client protein via a ubiquitin ligase (E3). Ubiquitination at the ER membrane can occur via cytoplasmic or ER-localized E3 ligases, both of which are shown. Retrotranslocation: for polytopic membrane proteins (pictured), retrotranslocation may occur by removal of the protein through a channel (retrotranslocon) and/or by removal of the protein and the surrounding membrane (not pictured). In either case, retrotranslocation almost always depends on the p97/Cdc48 complex, which includes Ufd1 and Npl4 and interacts with ubiquitin and misfolded regions on a substrate. p97/Cdc48 provides the mechanical force via ATP hydrolysis for substrate removal. Degradation: following retrotranslocation, misfolded proteins are ushered to the 26S proteasome and must be kept soluble to prevent aggregation. N-glycans are clipped by N-glycanse (not pictured), and ubiquitin moieties are removed by deubiquitinating enzymes either in the cytosol or in the proteasome cap. The proteasome contains three peptidase activities, trypsin-like, chymotrypsin-like, and caspase-like, which cleave proteins into short peptide fragments.