Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the ...cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill‐defined lysosomal catabolic pathways. Here, we describe an ER‐to‐lysosome‐associated degradation pathway (ERLAD) for proteasome‐resistant polymers of alpha1‐antitrypsin Z (ATZ). ERLAD involves the ER‐chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER‐resident ER‐phagy receptor FAM134B, echoing the initiation of starvation‐induced, receptor‐mediated ER‐phagy. However, in striking contrast to ER‐phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7‐positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single‐membrane, ER‐derived, ATZ‐containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER‐resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.
Synopsis
Misfolded proteins in the endoplasmic reticulum (ER) are dislocated across the ER membrane and degraded by the ubiquitin‐proteasome‐system. Proteasome‐resistant alpha1‐antitrypsin Z (ATZ) misfolded polymers undergo a novel ER‐to‐lysosome clearance pathway that requires ER‐phagy components, vesicular traffic and endolysosome fusion.
ATZ polymers are delivered from the ER to endolysosomal degradative compartments via receptor‐mediated vesicular traffic.
The ER‐chaperone Calnexin segregates ATZ polymers in ER subdomains and in ER‐derived vesicles under the control of ER‐phagy receptor FAM134B.
ATZ‐loaded vesicles recruit LC3 to dock and fuse with endolysosomes, leading to degradation of the ATZ polymers.
ER‐resident Syntaxin‐17 and lysosomal SNARE VAMP8 mediate membrane fusion events to guide allow degradation of misfolded polymers.
Clearance of proteasome‐resistant protein aggregates from the endoplasmic reticulum requires components of the LC3 lipidation machinery but occurs independently of autophagosome formation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Autophagy is a cytosolic quality control process that recognizes substrates through receptor‐mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the ...endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR‐Cas9 or knockout‐mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER‐resident lectin chaperone Calnexin (CANX) and the ER‐phagy receptor FAM134B are required for autophagy‐mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co‐receptor that recognizes ER luminal misfolded procollagens and interacts with the ER‐phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane‐associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome‐resistant misfolded clients from the ER.
Synopsis
Unfolded procollagen in the endoplasmic reticulum (ER) is an ER‐associated degradation‐resistant substrate that has to be cleared by autophagy. The ER chaperone Calnexin and the ER‐phagy receptor FAM134B recognize misfolded procollagen and mediate its LC3‐dependent delivery to the lysosome for autophagic degradation.
A candidate deletion screen shows that calnexin and FAM134B are required for ER quality control of endogenous procollagens
Calnexin acts as co‐receptor recognizing misfolded procollagen within the ER lumen
FAM134B binds misfolded procollagen through Calnexin and links it to LC3 on autophagosomal membranes
Calnexin and the ER‐phagy receptor FAM134B recognize misfolded procollagen in the ER and mediate its LC3‐dependent delivery to the lysosome for autophagic degradation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought ...to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The current SARS‐CoV‐2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID‐19 is why some individuals become sick and ...others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS‐CoV‐2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS‐CoV‐2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS‐CoV‐2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state‐of‐the‐art nucleic acid sequencing technologies, we can follow in detail how SARS‐CoV‐2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS‐CoV‐2 variants across the globe should be of key interest in our fight against the pandemic.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The unfolded protein response (UPR) is a cellular homeostatic circuit regulating protein synthesis and processing in the ER by three ER-to-nucleus signaling pathways. One pathway is triggered by the ...inositol-requiring enzyme 1 (IRE1), which splices the X-box binding protein 1 (
) mRNA, thereby enabling expression of XBP1s. Another UPR pathway activates the activating transcription factor 6 (ATF6). Here we show that murine cytomegalovirus (MCMV), a prototypic β-herpesvirus, harnesses the UPR to regulate its own life cycle. MCMV activates the IRE1-XBP1 pathway early post infection to relieve repression by XBP1u, the product of the unspliced
mRNA. XBP1u inhibits viral gene expression and replication by blocking the activation of the viral major immediate-early promoter by XBP1s and ATF6. These findings reveal a redundant function of XBP1s and ATF6 as activators of the viral life cycle, and an unexpected role of XBP1u as a potent repressor of both XBP1s and ATF6-mediated activation.
Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly ...investigated, since in most studies the ER-stress that is elicited cannot be overcome. Here we show that a proteostatic insult, achieved by persistent expression of the secretory heavy chain of immunoglobulin M (µ
), is well-tolerated in HeLa cells. Upon µ
expression, its levels temporarily eclipse those of the ER-chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER-stress in a ratiometric fashion. In the process the ER expands about threefold and becomes dominated by BiP. Since the UPR is essential for successful ER homeostatic readjustment in the HeLa-µ
model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER-stress.
The unfolded protein response (UPR) is one of the major cell‐autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore ...actively investigated as therapeutic target. In this respect, cell non‐autonomous effects of the UPR including the reported cell‐to‐cell transmission of UPR activity may be highly important. A pharmaca‐based UPR induction was employed to generate conditioned media (CM) from CM‐donating neuronal (‘donor’) cells (SK‐N‐SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal ‘acceptor’ cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry‐over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co‐culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry‐over of pharmaca is a major confounding factor in pharmaca‐based UPR transmission protocols that are therefore unsuitable to study cell‐to‐cell UPR transmission. In addition, the absence of UPR transmission in non‐pharmaca‐based models of UPR activation indicates that cell‐to‐cell UPR transmission does not occur in cell culture.
The unfolded protein response (UPR) is a major proteostatic stress response that was reported to be transmitted from cell‐to‐cell. Pharmacological induction of the UPR in ‘donor’ cells results in UPR activation in ‘acceptor’ cells via transfer of conditioned media. However, we show that carry‐over of pharmaca is a major confounding factor in this UPR transmission paradigm. Therefore, we employed multiple non‐pharmacological UPR transmission assays. Our data demonstrate that cell‐to‐cell transmission of UPR activity does not occur in cell culture.
Open Science: This manuscript was awarded with the Open Materials Badge
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible ...bulk expression of secretory immunoglobulin M heavy chain (μ
) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µ
is restored lead to µ
-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (C
1) from µ
. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem.
ABSTRACT
The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is ...special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures.
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BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely ...assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.
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•ERp44 governs a pH-regulated assembly control cycle in the early secretory pathway•Accessibility of ERp44’s active site and –RDEL ER retrieval motif is pH dependent•Unmasking of ERp44’s active site likely involves protonation of cysteine 29•ERp44 captures client proteins at cisGolgi-equivalent pH for retrieval to the ER
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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