The endoplasmic reticulum (ER) has a quality-control system for 'proof-reading' newly synthesized proteins, so that only native conformers reach their final destinations. Non-native conformers and incompletely assembled oligomers are retained, and, if misfolded persistently, they are degraded. As a large fraction of ER-synthesized proteins fail to fold and mature properly, ER quality control is important for the fidelity of cellular functions. Here, we discuss recent progress in understanding the conformation-specific sorting of proteins at the level of ER retention and export.

Quality control in the endoplasmic reticulum

Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-associated degradation (ERAD) pathway. During ERAD, molecular chaperones and associated factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin-proteasome machinery. The discovery of diseases that are associated with ERAD substrates highlights the importance of this pathway. Here, we summarize our current understanding of each step during ERAD, with emphasis on the factors that catalyse distinct activities.

One step at a time: endoplasmic reticulum-associated degradation

Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells activate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and translational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorder, which are collectively known as 'conformational diseases'. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2007.05639.x

ER stress and diseases

In homeostasis, cellular processes are in a dynamic equilibrium. Perturbation of homeostasis causes stress. In this review I summarize how perturbation of three major functions of the endoplasmic reticulum (ER) in eukaryotic cells–protein folding, lipid and sterol biosynthesis, and storing intracellular Ca2+ – causes ER stress and activates signaling pathways collectively termed the unfolded protein response (UPR). I discuss how the UPR reestablishes homeostasis, and summarize our current understanding of how the transition from protective to apoptotic UPR signaling is controlled, and how the UPR induces inflammatory signaling.

Endoplasmic reticulum stress responses.

A substantial fraction of eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) membrane. These polypeptides enter cotranslationally in the ER lumen, which contains resident molecular chaperones and folding factors that assist their maturation. Native proteins are released from the ER lumen and are transported through the secretory pathway to their final intra- or extracellular destination. Folding-defective polypeptides are exported across the ER membrane into the cytosol and destroyed. Cellular and organismal homeostasis relies on a balanced activity of the ER folding, quality control, and degradation machineries as shown by the dozens of human diseases related to defective maturation or disposal of individual polypeptides generated in the ER.

/pdf/in-and-out-of-the-er-protein-folding-quality-control-2dxm93bkk5.pdf

In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases

BACE457 is a recently identified pancreatic isoform of human β-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.

/pdf/sequential-assistance-of-molecular-chaperones-and-transient-23v115lu7j.pdf

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vascular endothelial growth factors (VEGFs). VEGF family proteins are produced in multiple isoforms with distinct biological properties and bind to three types of VEGF receptors. A VEGF-A splice variant, VEGF-A(165)b, has recently been isolated from kidney epithelial cells. This variant is identical to VEGF-A(165) except for the last six amino acids encoded by an alternative exon. VEGF-A(165)b and VEGF-A(165) bind VEGF receptors 1 and 2 with similar affinity. VEGF-A(165)b elicits drastically reduced activity in angiogenesis assays and even counteracts signaling by VEGF-A(165). VEGF-A(165)b weakly binds to heparan sulfate and does not interact with neuropilin-1, a coreceptor for VEGF receptor 2. To determine the molecular basis for altered signaling by VEGF-A(165)b we measured VEGF receptor 2 and ERK kinase activity in endothelial cells in culture. VEGF-A(165) induced strong and sustained activation of VEGF receptor 2 and ERK-1 and -2, while activation by VEGF-A(165)b was only weak and transient. Taken together these data show that VEGF-A(165)b has attenuated signaling potential through VEGF receptor 2 defining this new member of the VEGF family as a partial receptor agonist.

/pdf/a-vegf-a-splice-variant-defective-for-heparan-sulfate-and-2awcrks8wt.pdf

A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2.

The use of calnexin and calreticulin by cellular and viral glycoproteins.

Crystal structure of the orf virus NZ2 variant of vascular endothelial growth factor-E : Implications for receptor specificity

The crystal structure of VEGF-E was solved by the sulfur single-wavelength anomalous dispersion method (S-SAD) using highly redundant low-resolution data collected at a wavelength of lambda approximately 1.7 A with an estimated anomalous signal of 1.5%. 11 sulfur sites, nine out of 16 disulfide bonds and two out of 12 methionines could be located in the asymmetric unit using data truncated at a resolution of 4.1 A; however, none of the common diffraction data-quality indicators for SAD allowed clear discrimination between successful and unsuccessful resolution cutoffs. The high solvent content of 75% allowed efficient density modification to be performed and an unbiased electron-density map of good quality to be generated. This study demonstrates the strength of S-SAD for phasing using accurate highly redundant data at low resolution.

/pdf/structure-determination-of-vegf-e-by-sulfur-sad-rs6cdin8m7.pdf

Michel Pieren

Papers

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2.

The use of calnexin and calreticulin by cellular and viral glycoproteins.

Crystal structure of the orf virus NZ2 variant of vascular endothelial growth factor-E : Implications for receptor specificity

Structure determination of VEGF-E by sulfur SAD.