Zinc regulates ERp44-dependent protein quality control in the early secretory pathway.
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ABSTRACT:
SUBMITTER: Watanabe S
PROVIDER: S-EPMC6363758 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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Project description:ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.
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Project description:Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.
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Project description:Protein folding is inherently error prone, especially in the endoplasmic reticulum (ER). Even with an elaborate network of molecular chaperones and protein folding facilitators, misfolding can occur quite frequently. To maintain protein homeostasis, eukaryotes have evolved a series of protein quality-control checkpoints. When secretory pathway quality-control pathways fail, stress response pathways, such as the unfolded protein response (UPR), are induced. In addition, the ER, which is the initial hub of protein biogenesis in the secretory pathway, triages misfolded proteins by delivering substrates to the proteasome or to the lysosome/vacuole through ER-associated degradation (ERAD) or ER-phagy. Some misfolded proteins escape the ER and are instead selected for Golgi quality control. These substrates are targeted for degradation after retrieval to the ER or delivery to the lysosome/vacuole. Here, we discuss how these guardian pathways function, how their activities intersect upon induction of the UPR, and how decisions are made to dispose of misfolded proteins in the secretory pathway.
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Project description:ObjectiveLoss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of beta-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient beta-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes.Research design and methodsER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2(+/Akita) mutant mice were used as a model system to test the role of PERK in ERAD.ResultsWe report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes.ConclusionsPERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting beta-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.
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Project description:Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues.
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Project description:Serotonin N-acetyltransferase 1 (SNAT1), the penultimate enzyme for melatonin biosynthesis has shown N-acetyltransferase activity toward multiple substrates, including histones, serotonin, and plastid proteins. Under two different light conditions such as 50 or 100 μmol m-2 s-1, a SNAT1-knockout (snat1) mutant of Arabidopsis thaliana ecotype Columbia (Col-0) exhibited small size phenotypes relative over wild-type (WT) Arabidopsis Col-0. Of note, the small phenotype is stronger when growing at the 50 μmol m-2 s-1, exhibiting a dwarfism phenotype and delayed flowering. The snat1 Arabidopsis Col-0 accumulated less starch than the WT Col-0. Moreover, snat1 exhibited lower Lhcb1, Lhcb4, and RBCL protein levels, compared with the WT Col-0, but no changes in the corresponding transcripts, suggesting the involvement of melatonin in chloroplast protein quality control (CPQC). Accordingly, caseinolytic protease (Clp) and chloroplast heat shock proteins (CpHSPs), two key proteins involved in CPQC, as well as ROS defense were suppressed in snat1. In contrast, exogenous melatonin treatment induced expression of Clp, CpHSP, APX1, and GST, but not other growth-related genes such as DWF4, KS, and IAA1. Finally, the induction of ClpR1, APX1, and GST1 in response to melatonin was inhibited in the mitogen-activated protein kinase (MAPK) knockdown Arabidopsis (mpk3/6), suggesting that melatonin-mediated CPQC was mediated, in part, by the MAPK signaling cascade. These results suggest that melatonin is involved in CPQC, which plays a pivotal role in starch synthesis in plants.
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Project description:Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.
| S-EPMC4199500 | biostudies-literature
Project description:More than one-third of newly synthesized proteins are targeted to the early secretory pathway, which is comprised of the endoplasmic reticulum (ER), Golgi apparatus, and other intermediate compartments. The early secretory pathway plays a key role in controlling the folding, assembly, maturation, modification, trafficking, and degradation of such proteins. A considerable proportion of the secretome requires zinc as an essential factor for its structural and catalytic functions, and recent findings reveal that zinc plays a pivotal role in the function of the early secretory pathway. Hence, a disruption of zinc homeostasis and metabolism involving the early secretory pathway will lead to pathway dysregulation, resulting in various defects, including an exacerbation of homeostatic ER stress. The accumulated evidence indicates that specific members of the family of Zn transporters (ZNTs) and Zrt- and Irt-like proteins (ZIPs), which operate in the early secretory pathway, play indispensable roles in maintaining zinc homeostasis by regulating the influx and efflux of zinc. In this review, the biological functions of these transporters are discussed, focusing on recent aspects of their roles. In particular, we discuss in depth how specific ZNT transporters are employed in the activation of zinc-requiring ectoenzymes. The means by which early secretory pathway functions are controlled by zinc, mediated by specific ZNT and ZIP transporters, are also subjects of this review.
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Project description:Protein quality control in the early secretory pathway is a ubiquitous eukaryotic mechanism for adaptation to endoplasmic reticulum (ER) stress. An ER molecular chaperone, immunoglobulin heavy chain-binding protein (BiP), is one of the essential components in this process. BiP interacts with nascent proteins to facilitate their folding. BiP also plays an important role in preventing aggregation of misfolded proteins and regulating the ER stress response when cells suffer various injuries. BiP is a member of the 70-kDa heat shock protein (HSP70) family of molecular chaperones that resides in the ER. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity, leading to protein folding and maturation. BiP also possesses a retrieval motif in its carboxyl terminal. When BiP is secreted from the ER, the Lys-Asp-Glu-Leu (KDEL) receptor in the post-ER compartments binds with the carboxyl terminal KDEL sequence of BiP and returns BiP to the ER via coat protein complex I (COPI) vesicular transport. Although yeast studies showed that BiP retrieval by the KDEL receptor is not essential in single cells, it is crucial for multicellular organisms, where some essential proteins require retrieval to facilitate folding and maturation. Experiments in knock-in mice expressing mutant BiP with the retrieval motif deleted revealed a unique role of BiP retrieval by the KDEL receptor in neuronal development and age-related neurodegeneration.
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Project description: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.
| S-EPMC3699783 | biostudies-literature