ER-associated Degradation Machinery Research Articles

AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

Hypomorphic human SEL1L and HRD1 variants uncouple multilayered ER-associated degradation machinery.

The suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) complex of the endoplasmic reticulum-associated degradation (ERAD) machinery is a key cellular proteostasis pathway. Although previous studies have shown ERAD as promoting the development and maintenance of many cell types in mice, its importance to human physiology remained undetermined. In two articles in this issue of the JCI, Qi and colleagues describe four biallelic hypomorphic SEL1L and HRD1 variants that were associated with neurodevelopment disorders, locomotor dysfunction, impaired immunity, and premature death in patients. These pathogenic SEL1L-HRD1 variants shine a light on the critical importance of ERAD in humans and pave the way for future studies dissecting ERAD mechanisms in specific cell types.

Elucidation of molecular mechanism of the unfolded protein response.

The endoplasmic reticulum (ER), where newly synthesized secretory and transmembrane proteins are folded and assembled, has the ability to discriminate folded proteins from unfolded proteins and controls the quality of synthesized proteins. Only correctly folded molecules are allowed to move along the secretory pathway, whereas unfolded proteins are retained in the ER.The ER contains a number of molecular chaperones and folding enzymes (ER chaperones hereafter), which assist productive folding of proteins, and therefore newly synthesized proteins usually gain correct tertiary and quaternary structures quite efficiently. Yet unfolded or misfolded proteins even after assistance of ER chaperones are retrotranslocated back to the cytosol, ubiquitinated and degraded by the proteasome. This disposal system is called ER-associated degradation (ERAD). Thus, the quality of proteins in the ER is ensured by two distinct mechanisms, productive folding and ERAD, which have opposite directions.Under a variety of conditions collectively termed ER stress, however, unfolded or misfolded proteins accumulate in the ER, which in turn activates ER stress response or Unfolded Protein Response (UPR). The UPR is mediated by transmembrane proteins in the ER, and three ER stress sensors/transducers, namely IRE1, PERK and ATF6, operates ubiquitously in mammals. Thanks to these signaling pathways, translation is generally attenuated to decrease the burden on the folding machinery; transcription of ER chaperones is induced to augment folding capacity; and transcription of components of ERAD machinery is induced to enhance degradation capacity, leading to maintenance of the homeostasis of the ER. If ER stress sustains, cells undergo to apoptosis.I will talk on the mechanism, evolution, and physiological importance of the UPR and ERAD as well as its involvement in development and progression of various diseases.

Herp regulates intracellular survival of Mycobacterium tuberculosis H37Ra in macrophages by regulating reactive oxygen species-mediated autophagy.

Several studies have suggested that endoplasmic reticulum (ER) stress is important in the pathogenesis of infectious diseases; however, the precise function of ER stress regulation and the role of Herp as a regulator in Mtb H37Ra-induced ER stress remain elusive. Therefore, our study investigated ER stress and autophagy associated with Herp expression in Mycobacterium tuberculosis-infected macrophages to determine the role of Herp in the pathogenesis of tuberculosis.

Association of endoplasmic reticulum associated degradation (ERAD) with the transmission of Liberibacter solanacearum by its psyllid vector.

Candidatus Liberibacter solanacearum (CLso) is a complex of gram negative plant pathogenic and fastidious bacterial haplotypes restricted to the phloem and transmitted by several psyllid species. In Israel, the carrot psyllid Bactericera trigonica transmits CLso haplotype D in a persistent and propagative manner and causes the carrot yellows disease, inflicting significant economic losses in many countries. Understanding the transmission of CLso is fundamental to devising sustainable management strategies. Persistent transmission of vector-borne pathogens involves the critical steps of adhesion, cell invasion and replication inside the insect gut cells before passage to the hemolymph. Using microscopy and expression analyses, we have previously confirmed a role for the endoplasmic reticulum (ER) in inducing immune responses and subsequent molecular pathways resulting in programmed cell death (apoptosis) upon CLso-infection in the midgut. In the current study, we confirm that the ER-associated degradation (ERAD) machinery and its associated marker genes were upregulated in CLso infected insects, including Derlin-1, Selenoprotein-1 and Ubiquitin Ligase RNF-185. Silencing Derlin-1, which acts on the ER membrane by regulating the degradation of unfolded proteins upon ER stress, revealed its role in CLso persistence and transmission. Molecular pathways initiated in the ER membrane upon bacterial infection are well documented in human, animal and insect systems, and this study confirms the role of the ER in CLso-psyllid interactions.

Endoplasmic reticulum associated degradation is essential for maintaining the viability or function of mature myelinating cells in adults.

Endoplasmic reticulum associated degradation (ERAD) is responsible for recognition and degradation of unfolded or misfolded proteins in the ER. Sel1L is essential for the ERAD activity of Sel1L-Hrd1 complex, the best-known ERAD machinery. Using a continuous Sel1L knockout mouse model (CNP/Cre; Sel1LloxP/loxP mice), our previous studies showed that Sel1L knockout in myelinating cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS), leads to adult-onset myelin abnormalities in the CNS and PNS. Because Sel1L is deleted in myelinating cells of CNP/Cre; Sel1LloxP/loxP mice starting at very early stage of differentiation, it is impossible to rule out the possibility that the adult-onset myelin abnormalities in these mice results from developmental myelination defects caused by Sel1L knockout in myelinating cells during development. Thus, using an inducible Sel1L knockout mouse model (PLP/CreERT ; Sel1LloxP/loxP mice) that has normal, intact myelin and myelinating cells in the adult CNS and PNS prior to tamoxifen treatment, we sought to determine if Sel1L knockout in mature myelinating cells of adult mice leads to myelin abnormalities in the CNS and PNS. We showed that Sel1L knockout in mature myelinating cells caused ERAD impairment, ER stress and UPR activation. Interesting, Sel1L knockout in mature oligodendrocytes impaired their myelinating function by suppressing myelin protein translation, and resulted in progressive myelin thinning in the adult CNS. Conversely, Sel1L knockout in mature Schwann cells led to Schwann cell apoptosis and demyelination in the adult PNS. These findings demonstrate the essential roles of ERAD in mature myelinating cells in the adult CNS and PNS under physiological conditions.

An antisteatosis response regulated by oleic acid through lipid droplet-mediated ERAD enhancement.

Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here, we show that OA benefits Caenorhabditis elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet-dependent enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal an unexpected mechanism of FA signal transduction, as well as a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate some benefits of the OA-rich Mediterranean diet.

Abstract 11616: Obstructive Sleep Apnea Disrupts Endothelial Cellular Cholesterol Trafficking

Introduction: Obstructive sleep apnea (OSA), a highly prevalent disorder, triples cardiovascular risk. Our previous studies showed that intermittent hypoxia (IH), the hallmark of OSA, triggers endothelial cell (EC) inflammation by reducing protection against complement activity, which is cholesterol-dependent. We assessed whether IH promotes accumulation of cellular cholesterol in ECs in OSA patients and investigated the underlying mechanisms. Methods: We used ECs collected from OSA patients and cultured human umbilical vein ECs (HUVECs) exposed to IH (alternating 30 min 21% O 2 for normoxia/30 min 2% O 2 for hypoxia for 8 h). Results: Levels of free, cellular cholesterol were increased and the presence of lipid droplets was reduced in ECs from OSA patients compared with OSA-free controls as well as in HUVECs exposed to IH compared with normoxia. Cholesterol uptake and synthesis were similar in normoxia and IH. In contrast, co-localization of free cholesterol and late endosome/lysosome (LE/LY) was increased in IH compared with normoxia, suggesting impaired trafficking of free cholesterol from LE/LY to endoplasmic reticulum (ER) in IH. Immunoglobulin binding protein (BiP), a marker of ER stress, was upregulated in ECs of OSA patients compared with controls. IH-induced ER stress reduced interaction between LE/LY-bound oxysterol binding protein-related protein-1L (ORP1L) and ER-bound VAMP-associated protein-B (VAPB), which is required for cholesterol trafficking from LE/LY to ER. After screening ER-associated degradation machinery components for potential binding to VAPB in IH, we identified Derlin1 as a ligand that binds increasingly to VAPB in IH thereby potentially reducing interaction between ORP1L and VAPB. In OSA patients, ORP1L-VAPB interaction was reduced whereas Derlin1-VAPB was increased and positive airway pressure, a standard therapy for OSA, reversed these changes. Conclusions: IH induces ER stress that disrupts cholesterol trafficking from LE/LY to ER leading to accumulation of free cholesterol in ECs, which underlies impaired endothelial protection against complement and endothelial inflammation that may increase cardiovascular risk in OSA.

Characterization of dietary and herbal sourced natural compounds that modulate SEL1L-HRD1 ERAD activity and alleviate protein misfolding in the ER

Dysregulated production of peptide hormones is the key pathogenic factor of various endocrine diseases. Endoplasmic reticulum (ER) associated degradation (ERAD) is a critical machinery in maintaining ER proteostasis in mammalian cells by degrading misfolded proteins. Dysfunction of ERAD leads to maturation defect of many peptide hormones, such as provasopressin (proAVP), which results in the occurrence of Central Diabetes Insipidus. However, drugs targeting ERAD to regulate the production of peptide hormones are very limited. Herbal products provide not only nutritional sources, but also alternative therapeutics for chronic diseases. Virtual screening provides an effective and high-throughput strategy for identifying protein structure-based interacting compounds extracted from a variety of dietary or herbal sources, which could be served as (pro)drugs for preventing or treating endocrine diseases. Here, we performed a virtual screening by directly targeting SEL1L of the most conserved SEL1L-HRD1 ERAD machinery. Further, we analyzed 58 top-ranked compounds and demonstrated that Cryptochlorogenic acid (CCA) showed strong affinity with the binding pocket of SEL1L with HRD1. Through structure-based docking, protein expression assays, and FACS analysis, we revealed that CCA enhanced ERAD activity and promoted the degradation of misfolded proAVP, thus facilitated the secretion of well-folded proAVP. These results provide us with insights into drug discovery strategies targeting ER protein homeostasis, as well as candidate compounds for treating hormone-related diseases.

Regulation and Functions of the ER-Associated Nrf1 Transcription Factor.

Nrf1 is a member of the nuclear erythroid 2-like family of transcription factors that regulate stress-responsive gene expression in animals. Newly synthesized Nrf1 is targeted to the endoplasmic reticulum (ER) where it is N-glycosylated. N-glycosylated Nrf1 is trafficked to the cytosol by the ER-associated degradation (ERAD) machinery and is subject to rapid proteasomal degradation. When proteasome function is impaired, Nrf1 escapes degradation and undergoes proteolytic cleavage and deglycosylation. Deglycosylation results in deamidation of N-glycosylated asparagine residues to edit the protein sequence encoded by the genome. This truncated and "sequence-edited" form of Nrf1 enters the nucleus where it induces up-regulation of proteasome subunit genes. Thus, Nrf1 drives compensatory proteasome biogenesis in cells exposed to proteasome inhibitor drugs and other proteotoxic insults. In addition to its role in proteasome homeostasis, Nrf1 is implicated in responses to oxidative stress, and maintaining lipid and cholesterol homeostasis. Here, we describe the conserved and complex mechanism by which Nrf1 is regulated and highlight emerging evidence linking this unusual transcription factor to development, aging, and disease.

Protein disulfide isomerase PDI-6 regulates Wnt secretion to coordinate inter-tissue UPRmt activation and lifespan extension in C.elegans.

Coordination of inter-tissue stress signaling is essential for organismal fitness. Neuronal mitochondrial perturbations activate the mitochondrial unfolded-protein response (UPRmt) in the intestine via the mitokine Wnt signaling in Caenorhabditis elegans. Here, we found that the protein disulfide isomerase PDI-6 coordinates inter-tissue UPRmt signaling via regulating the Wnt ligand EGL-20. PDI-6 is expressed in the endoplasmic reticulum (ER) and interacts with EGL-20 through disulfide bonds that are essential for EGL-20 stability and secretion. pdi-6 deficiency results in misfolded EGL-20, which leads to its degradation via ER-associated protein degradation (ERAD) machinery. Expression of PDI-6 declines drastically with aging, and animals with pdi-6 deficiency have decreased lifespan. Overexpression of PDI-6 is sufficient to maintain Wnt/EGL-20 protein levels during aging, activating the UPRmt, and significantly extending lifespan in a Wnt- and UPRmt-dependent manner. Our study reveals that protein disulfide isomerase facilitates Wnt secretion to coordinate the inter-tissue UPRmt signaling and organismal aging.

Coronavirus Usurps the Autophagy-Lysosome Pathway and Induces Membranes Rearrangement for Infection and Pathogenesis.

Autophagy is a crucial and conserved homeostatic mechanism for early defense against viral infections. Recent studies indicate that coronaviruses (CoVs) have evolved various strategies to evade the autophagy–lysosome pathway. In this minireview, we describe the source of double-membrane vesicles during CoV infection, which creates a microenvironment that promotes viral RNA replication and virion synthesis and protects the viral genome from detection by the host. Firstly, CoVs hijack autophagy initiation through non-structural proteins and open-reading frames, leading to the use of non-nucleated phagophores and omegasomes for autophagy-derived double-membrane vesicles. Contrastingly, membrane rearrangement by hijacking ER-associated degradation machinery to form ER-derived double-membrane vesicles independent from the typical autophagy process is another important routine for the production of double-membrane vesicles. Furthermore, we summarize the molecular mechanisms by which CoV non-structural proteins and open-reading frames are used to intercept autophagic flux and thereby evade host clearance and innate immunity. A comprehensive understanding of the above mechanisms may contribute to developing novel therapies and clinical drugs against coronavirus disease 2019 (COVID-19) in the future.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway

ABSTRACT Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection. Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: β-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

SLC26A9 is selected for endoplasmic reticulum associated degradation (ERAD) via Hsp70-dependent targeting of the soluble STAS domain.

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis. To this end, we developed a novel yeast expression system for SLC26A9. This facile system has been used extensively with other ion channels and transporters to screen for factors that oversee protein folding checkpoints. As commonly observed for other channels and transporters, we first noted that a substantial fraction of SLC26A9 is targeted for endoplasmic reticulum associated degradation (ERAD), which destroys folding-compromised proteins in the early secretory pathway. We next discovered that ERAD selection requires the Hsp70 chaperone, which can play a vital role in ERAD substrate selection. We then created SLC26A9 mutants and found that the transmembrane-rich domain of SLC26A9 was quite stable, whereas the soluble cytosolic STAS domain was responsible for Hsp70-dependent ERAD. To support data obtained in the yeast model, we were able to recapitulate Hsp70-facilitated ERAD of the STAS domain in human tissue culture cells. These results indicate that a critical barrier to nascent membrane protein folding can reside within a specific soluble domain, one that is monitored by components associated with the ERAD machinery.

A plant reovirus hijacks the DNAJB12-Hsc70 chaperone complex to promote viral spread in its planthopper vector.

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus‐encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black‐streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus‐containing tubules composed of nonstructural membrane protein P7‐1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER‐associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co‐chaperone Hsc70, are activated by SRBSDV to facilitate ER‐to‐cytosol export of P7‐1 tubules in S. furcifera. Both P7‐1 of SRBSDV and Hsc70 directly bind to the J‐domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7‐1, but Hsc70 overexpression promotes the transport of P7‐1 from the ER to the cytosol to initiate tubule assembly. Thus, P7‐1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7‐1 tubule assembly, suggesting that the proper folding and assembly of P7‐1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7‐1 tubules in virus‐infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7‐1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER‐to‐cytosol transport of P7‐1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.

The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells

Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.

The Impact of Endoplasmic Reticulum-Associated Protein Modifications, Folding and Degradation on Lung Structure and Function.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and induces the unfolded protein response (UPR) and other mechanisms to restore ER homeostasis, including translational shutdown, increased targeting of mRNAs for degradation by the IRE1-dependent decay pathway, selective translation of proteins that contribute to the protein folding capacity of the ER, and activation of the ER-associated degradation machinery. When ER stress is excessive or prolonged and these mechanisms fail to restore proteostasis, the UPR triggers the cell to undergo apoptosis. This review also examines the overlooked role of post-translational modifications and their roles in protein processing and effects on ER stress and the UPR. Finally, these effects are examined in the context of lung structure, function, and disease.

A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions.

In the field, many insect-borne crop viral diseases are more suitable for maintenance and spread in hot-temperature areas, but the mechanism remains poorly understood. The epidemic of a planthopper (Sogatella furcifera)-transmitted rice reovirus (southern rice black-streaked dwarf virus, SRBSDV) is geographically restricted to southern China and northern Vietnam with year-round hot temperatures. Here, we reported that two factors of endoplasmic reticulum-associated degradation (ERAD) machinery, the heat shock protein DnaJB11 and ER membrane protein BAP31, were activated by viral infection to mediate the adaptation of S. furcifera to high temperatures. Infection and transmission efficiencies of SRBSDV by S. furcifera increased with the elevated temperatures. We observed that high temperature (35°C) was beneficial for the assembly of virus-containing tubular structures formed by nonstructural protein P7-1 of SRBSDV, which facilitates efficient viral transmission by S. furcifera. Both DnaJB11 and BAP31 competed to directly bind to the tubule protein P7-1 of SRBSDV; however, DnaJB11 promoted whereas BAP31 inhibited P7-1 tubule assembly at the ER membrane. Furthermore, the binding affinity of DnaJB11 with P7-1 was stronger than that of BAP31 with P7-1. We also revealed that BAP31 negatively regulated DnaJB11 expression through their direct interaction. High temperatures could significantly upregulate DnaJB11 expression but inhibit BAP31 expression, thereby strongly facilitating the assembly of abundant P7-1 tubules. Taken together, we showed that a new temperature-dependent protein quality control pathway in the ERAD machinery has evolved for strong activation of DnaJB11 for benefiting P7-1 tubules assembly to support efficient transmission of SRBSDV in high temperatures. We thus deduced that ERAD machinery has been hitchhiked by insect-borne crop viruses to enhance their transmission in tropical climates.

Regulation of hepatic circadian metabolism by the E3 ubiquitin ligase HRD1-controlled CREBH/PPARα transcriptional program

ObjectiveThe endoplasmic reticulum (ER)-resident E3 ligase HRD1 and its co-activator Sel1L are major components of ER-associated degradation (ERAD) machinery. Here, we investigated the molecular mechanism and functional significance underlying the circadian regulation of HRD1/Sel1L-mediated protein degradation program in hepatic energy metabolism. MethodsGenetically engineered animal models as well as gain- and loss-of-function studies were employed to address the circadian regulatory mechanism and functional significance. Gene expression, transcriptional activation, protein–protein interaction, and animal metabolic phenotyping analyses were performed to dissect the molecular network and metabolic pathways. ResultsHepatic HRD1 and Sel1L expression exhibits circadian rhythmicity that is controlled by the ER-tethered transcriptional activator CREBH, the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), and the core clock oscillator BMAL1 in mouse livers. HRD1/Sel1L mediates polyubiquitination and degradation of the CREBH protein across the circadian cycle to modulate rhythmic expression of the genes encoding the rate-limiting enzymes or regulators in fatty acid (FA) oxidation, triglyceride (TG) lipolysis, lipophagy, and gluconeogenesis. HRD1 liver-specific knockout (LKO) mice displayed increased expression of the genes involved in lipid and glucose metabolism and impaired circadian profiles of circulating TG, FA, and glucose due to overproduction of CREBH. The circadian metabolic activities of HRD1 LKO mice were inversely correlated with those of CREBH KO mice. Suppressing CREBH overproduction in the livers of HRD1 LKO mice restored the diurnal levels of circulating TG and FA of HRD1 LKO mice. ConclusionOur work revealed a key circadian-regulated molecular network through which the E3 ubiquitin ligase HRD1 and its co-activator Sel1L regulate hepatic circadian metabolism.

Protein Disulfide Isomerases Negatively Regulate Ebolavirus Structural Glycoprotein Expression

Ebola viruses (EBOV) express cytotoxic structural glycoprotein GP1,2 to mediate virus entry. To explore a novel viral fitness mechanism, we used mass spectrometry to investigate how EBOV-GP1,2 expression is regulated in the endoplasmic reticulum (ER). We found that ER protein 57 (ERp57), a member of protein disulfide isomerases (PDIs) that promote protein folding, targets EBOV-GP1,2 cysteine residues required for correct cross-linking, thus adverselytriggering misfolding, which in turn activates the unfolded protein response. Misfolded EBOV-GP1,2 is targeted by ER-associated protein degradation machinery and, unexpectedly, is degraded in lysosomes via autophagosomes, but not proteasomes. Five other PDIs also have similar inhibitory activity. In contrast, the expression of Marburg virus and some other enveloped virus glycoproteins is increased by ERp57. Thus, PDIs can negatively regulate EBOV glycoprotein expression, which enables optimum viral infectivity and minimizes cytotoxicity. Importantly, PDIs emerge as potential targets for inhibition of Ebola virus infection.