Endoplasmic Reticulum Quality Control Research Articles

The endoplasmic reticulum of trypanosomatids: An unrevealed road for chemotherapy.

The endoplasmic reticulum (ER) of higher eukaryotic cells forms an intricate membranous network that serves as the main processing facility for folding and assembling of secreted and membrane proteins. The ER is a highly dynamic organelle that interacts with other intracellular structures, as well as endosymbiotic pathogenic and non-pathogenic microorganisms. A strict ER quality control (ERQC) must work to ensure that proteins entering the ER are folded and processed correctly. Unfolded or misfolded proteins are usually identified, selected, and addressed to Endoplasmic Reticulum-Associated Degradation (ERAD) complex. Conversely, when there is a large demand for secreted proteins or ER imbalance, the accumulation of unfolded or misfolded proteins activates the Unfold Protein Response (UPR) to restore the ER homeostasis or, in the case of persistent ER stress, induces the cell death. Pathogenic trypanosomatids, such as Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp are the etiological agents of important neglected diseases. These protozoans have a complex life cycle alternating between vertebrate and invertebrate hosts. The ER of trypanosomatids, like those found in higher eukaryotes, is also specialized for secretion, and depends on the ERAD and non-canonical UPR to deal with the ER stress. Here, we reviewed the basic aspects of ER biology, organization, and quality control in trypanosomatids. We also focused on the unusual way by which T. cruzi, T. brucei, and Leishmania spp. respond to ER stress, emphasizing how these parasites' ER-unrevealed roads might be an attractive target for chemotherapy.

OR11-1 Thyroglobulin Structural Requirements in Protein Trafficking for Thyroid Hormonogenesis

Abstract In vertebrates, thyroglobulin (Tg) is secreted into the lumen of thyroid follicles to enable its iodination for the synthesis of thyroid hormone — defective Tg secretion causes hypothyroidism. A defining step in Tg secretion is export from the endoplasmic reticulum (ER), but Tg structural features needed to meet ER quality control requirements remain poorly defined. Tg primary structure is composed of Regions I, II, III, and ChEL, each engaged in intradomain disuflide bonding. Two major surprises that have recently emerged from the visualization of bovine Tg 3D-structure by cryoEM include the first identified long-range disulfide bond (C408-C608), which appears to tether a boundary separating two halves of region I, as well as the apparent absence of an evolutionarily conserved disulfide bond between C2444 and C2455 in the ChEL domain, which is known to support folding stability of region I. To understand the cell biological importance of these findings, we employed site-directed mutagenesis to determine the impact of these structural features in protein export of recombinant Tg expressed in cultured cells. To our surprise, we found that mutation of either a single Cys residue or both cysteines that contribute to the C408-C608 disulfide bridge had no discernible impact on the efficiency of Tg export. These data strongly suggest that the proximity of the two-halves of region I in the final Tg structure may be achieved by protein folding of individual repeating units of region I that must take place independently of (and thus presumably prior to) the formation of this interesting long-range disulfide bond. As ChEL supports Tg region I folding stability, we turned attention to the apparent absence of the C-2444-C2455 disulfide, which is one of the three disulfide bonds that had been thought to exist in every member of the large Cholinesterase-Like gene superfamily. Reported detection of this disulfide within human Tg, but lack of detection in bovine Tg, means either that this bond is 'structurally optional', or that this surface disulfide bond might have been broken by radiolysis under the X-ray beam used for cryoEM. Unequivocally, we found that mutagenesis of either C2444, C2455, or both, blocks Tg secretion and causes a pattern of intracellular misfolding consistent with incomplete Tg compaction indicative of lack of maturation of region I. Moreover, we found that disruption of these same residues causes misfolding and blocks secretion of an expressed isolated secretory ChEL domain, or acetylcholinesterase itself. Loss of the C2444-C2455 disulfide bridge behaves similarly to the defective folding observed in the mutant Tg encoded within cog/cog mice and rdw/rdw rats, whereas no pathogenic mutations have been reported affecting the long-range disulfide in Tg region I. Together, these results highlight the importance of ChEL domain structure in the maturation and secretion of Tg. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m.

The role of selective autophagy in pathogen infection

<p indent="0mm">Autophagy is a highly conserved physiological process of material degradation and recycling. According to the different pathways of degrading cargo, autophagy is classified into three types: Macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Macroautophagy refers to the process in which cargos to be degraded are wrapped in double-membrane autophagosome and transported to lysosomes for degradation. Microautophagy refers to the process in which lysosomal membrane invagination or protrusion wraps the cargo to be degraded and directly degrades it. Chaperone-mediated autophagy refers to the process in which molecular chaperones restore intracellular proteins from the folded state to the unfolded state, and then transport them to lysosomes for degradation. Autophagy was previously defined as a non-selective process induced under starvation conditions. However, developments in autophagy-related research and in-depth studies have revealed that autophagy can be either a non-selective or selective process. Selective autophagy refers to the selective cellular degradation process that targets and degrades dysfunctional organelles, misfolded proteins, or invading pathogens. According to the different targets of degradation, selective autophagy is divided into mitophagy, endoplasmic reticulum autophagy (ER-phagy), proteasome autophagy (proteaphagy), ribosomal autophagy (ribophagy), lipophagy, nucleophagy, and xenophagy, and so on. Currently, mitophagy, ER-phagy, and xenophagy are hot research topics. Mitophagy is an autophagic process that selectively targets and degrades damaged or redundant mitochondria and is essential for mitochondrial quality control and maintenance of cell homeostasis. In recent years, accumulating studies have shown that some viruses or bacteria regulate the mitophagy process by various direct or indirect strategies, thereby weakening innate immune responses such as type I interferon response, apoptosis, and the activation of inflammasome to facilitate infection. Further, ER-phagy is a selective autophagic process that targets and degrades damaged endoplasmic reticulum fragments and plays essential roles in endoplasmic reticulum quality control and maintenance of cellular homeostasis. The on-going studies on ER-phagy mechanism are limited compared with those on mitophagy mechanism, but studies have reported that ER-phagy plays a vital role in the immune escape of pathogens. Xenophagy refers to the process in which eukaryotic cells recognize and degrade invading pathogens through selective autophagy. However, some studies have reported that some invading pathogens such as <italic>Mycobacterium tuberculosis</italic> and <italic>Salmonella</italic> inhibit xenophagy in host to achieve intracellular survival. Selective autophagy is a process that targets different cargo that is to be degraded and shares some crucial autophagy-related proteins. When multiple selective autophagy processes simultaneously occur, these processes compete for the shared proteins. However, studies on the crosstalk between different selective autophagy processes are limited. In summary, this article reviews the molecular mechanisms of mitophagy, ER-phagy, and xenophagy and their roles in pathogen infection to provide a reference for selective autophagy-related studies.

Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation

Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.

Morphine disrupts macrophage functions even during HIV infection.

HIV-associated neurocognitive impairment (HIV-NCI) is a debilitating comorbidity that reduces quality of life in 15-40% of people with HIV (PWH) taking antiretroviral therapy (ART). Opioid use has been shown to increase neurocognitive deficits in PWH. Monocyte-derived macrophages (MDMs) harbor HIV in the CNS even in PWH on ART. We hypothesized that morphine (MOR), a metabolite of heroin, further dysregulates functional processes in MDMs to increase neuropathogenesis. We found that, in uninfected and HIV-infected primary human MDMs, MOR activates these cells by increasing phagocytosis and up-regulating reactive oxygen species. Effects of MOR on phagocytosis were dependent on μ-opioid receptor activity and were mediated, in part, by inhibited lysosomal degradation of phagocytized substrates. All results persisted when cells were treated with both MOR and a commonly prescribed ART cocktail, suggesting minimal impact of ART during opioid exposure. We then performed mass spectrometry in HIV-infected MDMs treated with or without MOR to determine proteomic changes that suggest additional mechanisms by which opioids affect macrophage homeostasis. Using downstream pathway analyses, we found that MOR dysregulates ER quality control and extracellular matrix invasion. Our data indicate that MOR enhances inflammatory functions and impacts additional cellular processes in HIV-infected MDMs to potentially increases neuropathogenesis in PWH using opioids.

SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

SummaryEndoplasmic reticulum (ER) homeostasis has been implicated in the pathogenesis of various forms of cancer; however, our understanding of the role of ER quality control mechanisms in tumorigenesis remains incomplete. Here, we show that the SEL1L-HRD1 complex of ER-associated degradation (ERAD) suppresses hepatocyte proliferation and tumorigenesis in mice. Hepatocyte-specific deletion of Sel1L or Hrd1 predisposed mice to diet/chemical-induced tumors. Proteomics screen from SEL1L-deficient livers revealed WNT5A, a tumor suppressor, as an ERAD substrate. Indeed, nascent WNT5A was misfolding prone and degraded by SEL1L-HRD1 ERAD in a quality control capacity. In the absence of ERAD, WNT5A misfolds is largely retained in the ER and forms high-molecular weight aggregates, thereby depicting a loss-of-function effect and attenuating WNT5A-mediated suppression of hepatocyte proliferation. In humans, SEL1L-HRD1 ERAD expression correlated positively with survival time for patients with liver cancer. Overall, our data reveal a key role of SEL1L-HRD1 ERAD in suppressing hepatocyte proliferation and liver cancer.

Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity

Adaptive immunity depends on cell surface presentation of antigenic peptides by major histocompatibility complex class I (MHC I) molecules and on stringent ER quality control in the secretory pathway. The chaperone tapasin in conjunction with the oxidoreductase ERp57 is crucial for MHC I assembly and for shaping the epitope repertoire for high immunogenicity. However, how the tapasin–ERp57 complex engages MHC I clients has not yet been determined at atomic detail. Here, we present the 2.7-Å crystal structure of a tapasin–ERp57 heterodimer in complex with peptide-receptive MHC I. Our study unveils molecular details of client recognition by the multichaperone complex and highlights elements indispensable for peptide proofreading. The structure of this transient ER quality control complex provides the mechanistic basis for the selector function of tapasin and showcases how the numerous MHC I allomorphs are chaperoned during peptide loading and editing.

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

The functional specificity of ERECTA-family receptors in Arabidopsis stomatal development is ensured by molecular chaperones in the endoplasmic reticulum.

Stomata are epidermal pores that control gas exchange between plants and the atmosphere. In Arabidopsis, the ERECTA family (ERECTAf) receptors, including ERECTA, ERECTA-LIKE 1 (ERL1) and ERL2, redundantly play pivotal roles in enforcing the 'one-cell-spacing' rule. Accumulating evidence has demonstrated that the functional specificities of receptors are likely associated with their differential subcellular dynamics. The endoplasmic reticulum (ER)-resident chaperone complex SDF2-ERdj3B-BiP functions in many aspects of plant development. We employed pharmacological treatments combined with cell biological and biochemical approaches to demonstrate that the abundance of ERECTA was reduced in the erdj3b-1 mutant, but the localization and dynamics of ERECTA were not noticeably affected. By contrast, the erdj3b mutation caused the retention of ERL1/ERL2 in the ER. Furthermore, we found that the function of SDF2-ERdj3B-BiP is implicated with the distinct roles of ERECTAf receptors. Our findings establish that the ERECTAf receptor-mediated signaling in stomatal development is ensured by the activities of the ER quality control system, which preferentially maintains the protein abundance of ERECTA and proper subcellular dynamics of ERL1/ERL2, prior to the receptors reaching their destination - the plasma membrane - to execute their functions.

Identification of two rate-limiting steps in the degradation of partially folded immunoglobulin light chains

Antibody monomers are produced from two immunoglobulin heavy chains and two light chains that are folded and assembled in the endoplasmic reticulum This process is assisted and monitored by components of the endoplasmic reticulum quality control machinery; an outcome made more fraught by the unusual genetic machinations employed to produce a seemingly unlimited antibody repertoire. Proper functioning of the adaptive immune system is as dependent on the success of this operation, as it is on the ability to identify and degrade those molecules that fail to reach their native state. In this study, two rate-limiting steps were identified in the degradation of a non-secreted κ light chain. Both focus on the constant domain (CL), which has evolved to fold rapidly and very stably to serve as a catalyst for the folding of the heavy chain CH1 domain. The first hurdle is the reduction of the disulfide bond in the CL domain, which is required for retrotranslocation to the cytosol. In spite of being reduced, the CL domain retains structure, giving rise to the second rate-limiting step, the unfolding of this domain at the proteasome, which results in a stalled degradation intermediate.

Molecular basis of MHC I quality control in the peptide loading complex

Major histocompatibility complex class I (MHC I) molecules are central to adaptive immunity. Their assembly, epitope selection, and antigen presentation are controlled by the MHC I glycan through a sophisticated network of chaperones and modifying enzymes. However, the mechanistic integration of the corresponding processes remains poorly understood. Here, we determine the multi-chaperone-client interaction network of the peptide loading complex (PLC) and report the PLC editing module structure by cryogenic electron microscopy at 3.7 Å resolution. Combined with epitope-proofreading studies of the PLC in near-native lipid environment, these data show that peptide-receptive MHC I molecules are stabilized by multivalent chaperone interactions including the calreticulin-engulfed mono-glucosylated MHC I glycan, which only becomes accessible for processing by α-glucosidase II upon loading of optimal epitopes. Our work reveals allosteric coupling between peptide-MHC I assembly and glycan processing. This inter-process communication defines the onset of an adaptive immune response and provides a prototypical example of the tightly coordinated events in endoplasmic reticulum quality control.

The testis-specific E3 ubiquitin ligase RNF133 is required for fecundity in mice

BackgroundUbiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown.ResultsWe identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133.ConclusionsOur studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts.

Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.

KDEL Receptors: Pathophysiological Functions, Therapeutic Options, and Biotechnological Opportunities.

KDEL receptors (KDELRs) are ubiquitous seven-transmembrane domain proteins encoded by three mammalian genes. They bind to and retro-transport endoplasmic reticulum (ER)-resident proteins with a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence or variants thereof. In doing this, KDELR participates in the ER quality control of newly synthesized proteins and the unfolded protein response. The binding of KDEL proteins to KDELR initiates signaling cascades involving three alpha subunits of heterotrimeric G proteins, Src family kinases, protein kinases A (PKAs), and mitogen-activated protein kinases (MAPKs). These signaling pathways coordinate membrane trafficking flows between secretory compartments and control the degradation of the extracellular matrix (ECM), an important step in cancer progression. Considering the basic cellular functions performed by KDELRs, their association with various diseases is not surprising. KDELR mutants unable to bind the collagen-specific chaperon heat-shock protein 47 (HSP47) cause the osteogenesis imperfecta. Moreover, the overexpression of KDELRs appears to be linked to neurodegenerative diseases that share pathological ER-stress and activation of the unfolded protein response (UPR). Even immune function requires a functional KDELR1, as its mutants reduce the number of T lymphocytes and impair antiviral immunity. Several studies have also brought to light the exploitation of the shuttle activity of KDELR during the intoxication and maturation/exit of viral particles. Based on the above, KDELRs can be considered potential targets for the development of novel therapeutic strategies for a variety of diseases involving proteostasis disruption, cancer progression, and infectious disease. However, no drugs targeting KDELR functions are available to date; rather, KDELR has been leveraged to deliver drugs efficiently into cells or improve antigen presentation.

The Rice Malectin Regulates Plant Cell Death and Disease Resistance by Participating in Glycoprotein Quality Control.

In animals, malectin is well known to play an essential role in endoplasmic reticulum quality control (ERQC) by interacting with ribophorin I, one unit of the oligosaccharyltransferase (OST) complex. However, the functions of malectin in plants remain largely unknown. Here, we demonstrate the rice OsMLD1 is an ER- and Golgi-associated malectin protein and physically interacts with rice homolog of ribophorin I (OsRpn1), and its disruption leads to spontaneous lesion mimic lesions, enhanced disease resistance, and prolonged ER stress. In addition, there are many more N-glycosites and N-glycoproteins identified from the mld1 mutant than wildtype. Furthermore, OsSERK1 and OsSERK2, which have more N-glycosites in mld1, were demonstrated to interact with OsMLD1. OsMLD1 can suppress OsSERK1- or OsSERK2-induced cell death. Thus, OsMLD1 may play a similar role to its mammalian homologs in glycoprotein quality control, thereby regulating cell death and immunity of rice, which uncovers the function of malectin in plants.

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.

Misfolded GBA/β-glucocerebrosidase impairs ER-quality control by chaperone-mediated autophagy in Parkinson disease

Inhibition of chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, may contribute to pathogenesis in neurodegenerative diseases including Parkinson disease (PD). Pathogenic variants of PD-related proteins that reside in the cytosol, including SNCA/alpha-synuclein, LRRK2 (leucine rich repeat kinase 2), UCHL1 (ubiquitin Cterminal hydrolase 1) and VPS35 (VPS35 retromer complex component), exert inhibitory effects on CMA. Decreased CMA activity has also been reported in sporadic PD patients, consistent with an association between CMA inhibition and PD. We have now reported the first example of CMA dysfunction caused by a non-cytosolic PD-related protein, GBA/β-glucocerebrosidase, the most common genetic risk factor for PD, which uncovers a new role for CMA in endoplasmic reticulum (ER) quality control.

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Tandem mass tag-based proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under ultraviolet-B and dark stress.

Mulberry leaves have been used in traditional Chinese medicine due to their antioxidant, antidiabetic, and antihyperlipidemic properties. A previous study showed that ultraviolet-B radiation followed by dark incubation could improve the contents of active ingredients in mulberry leaves, such as moracin N and chalcomoracin. The endoplasmic reticulum (ER) serves as a protein quality control center and the location for protein synthesis, which is involved in the response to the environmental stress in plants. To investigate the mechanisms in response to ultraviolet-B radiation followed by dark incubation (UV + D), ER proteomics was performed on mulberry leaves. The ER protein markers, glucose-regulated protein (GRP78), and calnexin (CNX), were significantly higher in the ER fraction than in the total protein fraction, indicating that the ER was purified. Compared to the control, the abundance of protein disulfide isomerase, UDP-glucose glycoprotein glucosyltransferase, CNX, and calreticulin proteins decreased, while of the abundance of heat shock-related proteins increased under stress. P450 enzyme system-related proteins and ribosomal proteins showed significant increases. These results suggest that under UV + D stress, mulberry leaves activated the cell redox and ER quality control systems, enhancing protein synthesis and weakening N-glycan biosynthesis in the ER to resist the damage.

A plant receptor domain with functional analogies to animal malectin disables ER stress responses upon infection

SummaryMalectins from the oligosaccharyltransferase (OST) complex in the endoplasmic reticulum (ER) of animal cells are involved in ER quality control and contribute to the Unfolded Protein Response (UPR). Malectins are not found in plant cells, but malectin-like domains (MLDs) are constituents of many membrane-bound receptors. In Arabidopsis thaliana, the MLD-containing receptor IOS1 promotes successful infection by filamentous plant pathogens. We show that the MLD of its exodomain retains IOS1 in the ER of plant cells and attenuates the infection-induced UPR. Expression of the MLD in the ios1-1 knockout background is sufficient to complement infection-related phenotypes of the mutant, such as increased UPR and reduced disease susceptibility. IOS1 interacts with the ER membrane-associated ribophorin HAP6 from the OST complex, and hap6 mutants show decreased pathogen-responsive UPR and increased disease susceptibility. Altogether, this study revealed a previously uncharacterized role of a plant receptor domain in the regulation of ER stress during infection.