Sorting,recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome

Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones.Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.     

IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.     

The oligomeric state of Derlin-1 is modulated by endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.     

Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways

The accumulation of aberrantly folded proteins can lead to cell dysfunction and death. Currently, the mechanisms of toxicity and cellular defenses against their effects remain incompletely understood. In the endoplasmic reticulum (ER), stress caused by misfolded proteins activates the unfolded protein response (UPR). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes to maintain cellular homeostasis. We studied the effects of ER stress in budding yeast through expression of the well-characterized misfolded protein, CPY*. By challenging cells within their physiological limits to resist stress, we show that the UPR is required to maintain essential functions including protein translocation, glycosylation, degradation, and transport. Under stress, the ER-associated degradation (ERAD) pathway for misfolded proteins is saturable. To maintain homeostasis, an "overflow" pathway dependent on the UPR transports excess substrate to the vacuole for turnover. The importance of this pathway was revealed through mutant strains compromised in the vesicular trafficking of excess CPY*. Expression of CPY* at levels tolerated by wild-type cells was toxic to these strains despite retaining the ability to activate the UPR.     

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

High ER stress in beta-cells stimulates intracellular degradation of misfolded insulin

Endoplasmic reticulum (ER) stress, which is caused by the accumulation of misfolded proteins in the ER, elicits an adaptive response, the unfolded protein response (UPR). One component of the UPR, the endoplasmic reticulum-associated protein degradation (ERAD) system, has an important function in the survival of ER stressed cells. Here, we show that HRD1, a component of the ERAD system, is upregulated in pancreatic islets of the Akita diabetes mouse model and enhances intracellular degradation of misfolded insulin. High ER stress in beta-cells stimulated mutant insulin degradation through HRD1 to protect beta-cells from ER stress and ensuing death. If HRD1 serves the same function in humans, it may serve as a target for therapeutic intervention in diabetes.     

The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca(2+) release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.     

RESETing ER proteostasis: selective stress pathway hidden in the secretory route

The efficient folding of membrane and secreted proteins relies on the unfolded protein response (UPR) to buffer fluctuations in the load of misfolded proteins. Although the UPR is thought to operate on a generic manner to maintain ER proteostasis, a recent study revealed the existence of a novel mechanism to eliminate misfolded GPI‐anchored proteins via the secretory pathway, termed ‘rapid ER stress‐induced export’ (RESET) (Satpute‐Krishnan et al, 2014 ). RESET involves the export of misfolded GPI proteins to the plasma membrane for subsequent degradation by the lysosome.     

BiP Availability Distinguishes States of Homeostasis and Stress in the Endoplasmic Reticulum of Living Cells

Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.     

A new role for a COPII cargo adaptor in autophagy

ABSTRACT

Endoplasmic reticulum (ER) homeostasis is maintained by the removal of misfolded ER proteins via different quality control pathways. Aggregation-prone proteins, including certain disease-linked proteins, are resistant to conventional ER degradation pathways and require other disposal mechanisms. Reticulophagy is a disposal pathway that uses resident autophagy receptors. How these receptors, which are dispersed throughout the ER network, target a specific ER domain for degradation is unknown. We recently showed in budding yeast, that ER stress upregulates the reticulophagy receptor, triggering its association with the COPII cargo adaptor complex, Sfb3/Lst1-Sec23 (SEC24C-SEC23 in mammals), to discrete sites on the ER. These domains are packaged into phagophores for degradation to prevent the accumulation of protein aggregates in the ER. This unconventional role for Sfb3/Lst1 is conserved in mammals and is independent of its role as a cargo adaptor on the secretory pathway. Our findings may have important therapeutic implications in protein-aggregation linked neurodegenerative disorders.     

Unfolded protein response

In eukaryotic cells, the endoplasmic reticulum (ER) is a membrane-enclosed interconnected organelle responsible for the synthesis, folding, modification, and quality control of numerous secretory and membrane proteins. The processes of protein folding and maturation are highly assisted and scrutinized but are also sensitive to changes in ER homeostasis, such as Ca(2+) depletion, oxidative stress, hypoxia, energy deprivation, metabolic stimulation, altered glycosylation, activation of inflammation, as well as increases in protein synthesis or the expression of misfolded proteins or unassembled protein subunits. Only properly folded proteins can traffic to the Golgi apparatus, whereas those that misfold are directed to ER-associated degradation (ERAD) or to autophagy. The accumulation of unfolded/misfolded proteins in the ER activates signaling events to orchestrate adaptive cellular responses. This unfolded protein response (UPR) increases the ER protein-folding capacity, reduces global protein synthesis, and enhances ERAD of misfolded proteins.     

Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.     

Autophagy: an ER protein quality control process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome-dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both alpha1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.     

UAE1 inhibition mediates the unfolded protein response,DNA damage and caspase-dependent cell death in pancreatic cancer

The Unfolded Protein Response (UPR) plays a key role in the adaptive response to loss of protein homeostasis within the endoplasmic reticulum (ER). The UPR has an adaptive function in protein homeostasis, however, sustained activation of the UPR due to hypoxia, nutrient deprivation, and increased demand for protein synthesis, alters the UPR program such that additional perturbation of ER homeostasis activates a pro-apoptotic program. Since ubiquitination followed by proteasomal degradation of misfolded proteins within the ER is a central mechanism for restoration of ER homeostasis, inhibitors of this pathway have proven to be valuable anti-cancer therapeutics. Ubiquitin activating enzyme 1(UAE1), activates ubiquitin for transfer to target proteins for proteasomal degradation in conjunction with E2 and E3 enzymes. Inhibition of UAE1 activity in response to TAK-243, leads to an accumulation of misfolded proteins within the ER, thereby aggravating ER stress, leading to DNA damage and arrest of cells in the G2/M phase of the cell cycle. Persistent drug treatment mediates a robust induction of apoptosis following a transient cell cycle arrest. These biological effects of TAK-243 were recapitulated in mouse models of PDAC demonstrating antitumor activity at a dose and schedule that did not exhibit obvious normal tissue toxicity. In vitro as well as studies in mouse models failed to show enhanced efficacy when TAK-243 was combined with ionizing radiation or gemcitabine, providing an impetus for future studies to identify agents that synergize with this class of agents for improved tumor control in PDAC.SignificanceThe UAE1 inhibitor TAK-243, mediates activation of the unfolded protein response, accumulation of DNA breaks and apoptosis, providing a rationale for the use as a safe and efficacious anti-cancer therapeutic for PDAC.     

JAMP optimizes ERAD to protect cells from unfolded proteins

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.