Paraoxonase 2 is an ER chaperone that regulates the epithelial Na+ channel

The mammalian paraoxonases have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2 and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in Fisher Rat Thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts prior to ENaC subunits ubiquitination. As a result of the enhanced ENaC subunits ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirect the channel for ER associated degradation.

Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.

Endoplasmic reticulum stress-induced release and binding of calreticulin from human ovarian cancer cells

Background Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). Methods We treated OC cells with the chemotherapeutic—doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor—thapsigargin (TG)—and/or an ER stress inhibitor—TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. Results Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. Conclusion Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

Explorations of ATP-Binding Cassette Transporters and Apoptosis Signal Pathways of 2-Hydroxyanthraquinone Substituted Cyclotriphosphazenes in MCF-7 and DLD-1 Cell Lines

Background: As a class with biological properties, such as anti-cancer, anti-bacterial, anti-HIV, and various physical effects, phosphazene derivatives constitute the most striking part of inorganic compounds. Anthraquinones, on the other hand, are a broad family of compounds with a wide variety of biological properties; the biologically active anthraquinones have been used as valuable tool compounds for biochemical and pharmacological research. Objective: In this study, we aimed to investigate the effect of the anthraquinone substituted cyclotriphosphazene compounds on apoptosis and drug resistance in MCF-7 and DLD-1 cells. Methods: In breast and colon cells, mRNA levels of multi-drug resistance genes (ABCB1, ABCC3, ABCC10, ABCC11, and ABCG2), apoptotic genes (BAX, BCL-2, p53, and PARP), heat shock (HSP27, HSP40, HSP60, HSP90α) and endoplasmic reticulum chaperone genes (GRP78, and GRP94) were determined by the qPCR method. The amount of proteins of the cell cycle, HSPs, apoptosis, and related signaling pathways were measured by the membrane array kits. Results: Compounds 2, 3, 4, and 7 showed the most potent results on the ATP-binding cassette genes in both breast and colon cancer cells. These compounds have a remarkable effect on apoptotic, heat shock, and ER chaperone genes in cancer cells. Besides, these compounds induced protein levels of pro-apoptotic pathways, leading to apoptosis by inhibiting anti-apoptotic pathways. Also, these compounds decreased HSPs. Conclusion: These compounds have potential properties that eliminate drug resistance, suppress heat shock and ER chaperone genes, and drag cells to apoptotic cell death and are notable for drug studies.

FK506-binding protein 2 (FKBP13) inhibit Bax-induced apoptosis in Saccharomyces cerevisiae (yeast)

FK506-binding protein 2 (FKBP13) is a part of the immunophilin protein family involved in immunoregulation. It is also believed to operate as a factor in membrane cytoskeletal framework and as an ER chaperone. FKBP2 (FKBP13) and FKBP1 (FKBP12), known as immunophilins, are binding proteins for rapamycin and FK506, which are immunosuppressive drugs. It was suggested that immunophilin-like and immunophilin proteins play significant roles in regulating intracellular calcium and protein folding/sorting, acting as molecular chaperones. Within the 15 mammalian FKBPs known, FKBP1 is merely the only one proven to form complexes with rapamycin and FK506 in the cytosol and facilitate their T cells immunosuppressive effects, FKBP2 is a luminal protein of the endoplasmic reticulum (ER) and is reported to take part in protein folding in the ER. However, little is known about FKBP2 link with apoptosis (either as a pro or anti-apoptotic protein). In this study, FKPB2 protein was co-expressed with the pro-apoptotic protein Bax after a yeast-based human hippocampal cDNA library screening. The yeast strain carrying the Bax gene was transformed with an episomal 2-micron plasmid that encodes the HA-tagged FKBP2 gene. The resultant strain would allow co-expression of Bax and FKBP2 in yeast cells. The results presented here show that a protein involved in protein folding can play a role in protecting yeast cell from Bax-induced apoptosis.

Ultrabright Plasmonic-Fluor Nanolabel-Enabled Detection of a Urinary ER Stress Biomarker in Autosomal Dominant Tubulointerstitial Kidney Disease

Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is the most common non-polycystic genetic kidney disease, but it remains unrecognized due to its clinical heterogeneity and lack of screening test. Moreover, clinical feature being a poor predictor of the disease outcome further highlights the need for development of mechanistic biomarkers in ADTKD. However, low abundant urinary proteins secreted by thick ascending limb (TAL) cells, where UMOD is synthesized, have posed a challenge on detection of biomarkers in ADTKD-UMOD. In the CRISPR/Cas9-generated murine model and patients with ADTKD-UMOD, we find that immunoglobulin heavy chain-binding protein (BiP), an ER chaperone, was exclusively upregulated by mutant UMOD in TAL and easily detected by Western blot in the urine at an early stage of disease. However, even the most sensitive ELISA failed to detect urinary BiP in affected individuals. We therefore developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA) to quantify urinary BiP concentration by harnessing the newly invented ultrabright fluorescent nanoconstruct, termed "plasmonic fluor" (Nat Biomed Eng 2020). p-FLISA demonstrated that urinary BiP excretion was significantly elevated in ADTKD-UMOD patients compared with unaffected controls, which may have potential utility in risk stratification, disease activity monitoring, disease progression prediction, and guidance of ER-targeted therapies in ADTKD.

Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.

Abstract P728: Promoting Neuron Survival After Ischemic Stroke via Restoring Astrocytic Endoplasmic Reticulum Function and Glucose Metabolism

Stroke-induced reactive astrocytes exhibit abnormal functions and contribute to neurodegeneration. Thus, restoring normal homeostatic astrocyte functions is important for neuroprotection. Overstimulation of Na + /H + exchanger 1 (NHE1) activity after stroke triggers reactive astrocyte formation, which displays abnormal functions. We have shown that targeted deletion of Nhe1 in astrocytes leads to inhibition of reactive astrocytes, reduced infarct volume, and improved neurological function after ischemic stroke. In the present study, we investigated ischemic stroke-mediated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) and its impact on cellular reparative functions. Astrocyte specific deletion of Nhe1 in Gfap-Cre ERT2+/- ;Nhe1 f/f mice ( Nhe1 Astro-KO) was induced by tamoxifen (Tam) treatment. Gfap-Cre ERT2- /- ;Nhe1 f/f mice treated with Tam served as wild-type (WT) controls. In transient middle cerebral artery occlusion (tMCAO) model, ischemic stroke triggered a significant increase in expression of ER stress marker proteins ATF4 and CHOP at 24 h reperfusion (Rp) in WT ischemic brains (p< 0.05), no such elevation was detected in Nhe1 Astro-KO ischemic brains. Immunocytochemical analysis revealed abundant ER chaperone protein GRP78 expression in non-ischemic cortical neurons (NeuN + ) of WT brains, but it was reduced by ~ 70% at 48 h Rp (p < 0.001). In contrast, Nhe1 Astro-KO brains preserved ER chaperone protein GRP78 in NeuN + cells in the ischemic cortex, and their adjacent GFAP + astrocytes showed ~40% increased GRP78 expression compared to WT. These polarized astrocytes with elevated GRP78 expression may provide neuroprotective support. Dysfunctional ER chaperone activity can affect glucose metabolism by decreasing glycolysis and mitochondrial respiration. Consistently, our bulk RNA-Seq analysis of reactive astrocytes shows that Nhe1 Astro-KO astrocytes exhibit significantly stimulated transcriptome profiles for glycolysis ( Hk2, Gpi, and Ldhb) and oxidative phosphorylation (complex I, IV and V). Together, our study demonstrates that restoring astrocytic ER chaperon function and glucose metabolism via specific deletion of Nhe1 promotes neuron survival after ischemic stroke.

The translocon-associated protein (TRAP) complex regulates quality control of N-linked glycosylation during ER stress

Asparagine (N)–linked glycosylation is required for endoplasmic reticulum (ER) homeostasis, but how this co- and posttranslational modification is maintained during ER stress is unknown. Here, we introduce a fluorescence-based strategy to detect aberrant N-glycosylation in individual cells and identify a regulatory role for the heterotetrameric translocon-associated protein (TRAP) complex. Unexpectedly, cells with knockout of SSR3 or SSR4 subunits restore N-glycosylation over time concurrent with a diminished ER stress transcriptional signature. Activation of ER stress or silencing of the ER chaperone BiP exacerbates or rescues the glycosylation defects, respectively, indicating that SSR3 and SSR4 enable N-glycosylation during ER stress. Protein levels of the SSR3 subunit are ER stress and UBE2J1 dependent, revealing a mechanism that coordinates upstream N-glycosylation proficiency with downstream ER-associated degradation and proteostasis. The fidelity of N-glycosylation is not static in both nontransformed and tumor cells, and the TRAP complex regulates ER glycoprotein quality control under conditions of stress.

Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.