COLEC10 Induces Endoplasmic Reticulum Stress by Occupying GRP78 and Inhibits Hepatocellular Carcinoma.

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker.

A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78.

Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.

AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases.

LRRK2 impairs autophagy by mediating phosphorylation of leucyl-tRNA synthetase.

Leucine-rich repeat kinase 2 (LRRK2) is a causal gene of Parkinson disease. G2019S pathogenic mutation increases its kinase activity. LRRK2 regulates various phenotypes including autophagy, neurite outgrowth, and vesicle trafficking. Leucyl-tRNA synthetase (LRS) attaches leucine to tRNALeu and activates mTORC1. Down-regulation of LRS induces autophagy. We investigated the relationship between LRRK2 and LRS in regulating autophagy and observed interaction between endogenous LRRK2 and LRS proteins and LRS phosphorylation by LRRK2. Mutation studies implicated that T293 in the LRS editing domain was a putative phosphorylation site. Phospho-Thr in LRS was increased in cells overexpressing G2019S and dopaminergic neurons differentiated from induced pluripotent stem (iPS) cells of a G2019S carrier. It was decreased by treatment with an LRRK2 kinase inhibitor (GSK2578215A). Phosphomimetic T293D displayed lower leucine bindings than wild type (WT), suggesting its defective editing function. Cellular expression of T293D increased expression of GRP78/BiP, LC3B-II, and p62 proteins and number of LC3 puncta. Increase of GRP78 and phosphorylated LRS was diminished by treatment with GSK2578215A. Levels of LC3B, GRP78/BiP, p62, and α-synuclein proteins were also increased in G2019S transgenic (TG) mice. These data suggest that LRRK2-mediated LRS phosphorylation impairs autophagy by increasing protein misfolding and endoplasmic reticulum stress mediated by LRS editing defect. SIGNIFICANCE OF THE STUDY: Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of Parkinson disease (PD), and the most prevalent pathogenic mutation, G2019S, increases its kinase activity. In this study, we elucidated that leucyl-tRNA synthetase (LRS) was an LRRK2 kinase substrate and identified T293 as an LRRK2 phosphorylation site. LRRK2-meidated LRS phosphorylation or G2019S can lead to impairment of LRS editing, increased ER stress, and accumulation of autophagy markers. These results demonstrate that LRRK2 kinase activity can facilitate accumulation of misfolded protein, suggesting that LRRK2 kinase might be a potential PD therapeutic target along with previous studies.

Characterization and mechanism of stress-induced translocation of 78-kilodalton glucose-regulated protein (GRP78) to the cell surface.

Glucose-regulated protein (GRP78)/BiP, a major chaperone in the endoplasmic reticulum, is recently discovered to be preferably expressed on the surface of stressed cancer cells, where it regulates critical oncogenic signaling pathways and is emerging as a target for anti-cancer therapy while sparing normal organs. However, because GRP78 does not contain classical transmembrane domains, its mechanism of transport and its anchoring at the cell surface are poorly understood. Using a combination of biochemical, mutational, FACS, and single molecule super-resolution imaging approaches, we discovered that GRP78 majorly exists as a peripheral protein on plasma membrane via interaction with other cell surface proteins including glycosylphosphatidylinositol-anchored proteins. Moreover, cell surface GRP78 expression requires its substrate binding activity but is independent of ATP binding or a membrane insertion motif conserved with HSP70. Unexpectedly, different cancer cell lines rely on different mechanisms for GRP78 cell surface translocation, implying that the process is cell context-dependent.

Unfolded protein response is activated in Lewy body dementias.

AIM: The unfolded protein response (UPR) is a pro-survival defence mechanism induced during periods of endoplasmic reticulum stress, and it has recently emerged as an attractive therapeutic target across a number of neurodegenerative conditions, but has not yet been studied in synuclein disorders. METHODS: The level of a key mediator of the UPR pathway, glucose-regulated protein 78 (GRP78), also known as binding immunoglobulin protein (BiP), was measured in post mortem brain tissue of patients with dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) in comparison with Alzheimer's disease (AD) and age-matched controls using Western blot. The UPR activation was further confirmed by immunohistochemical detection of GRP78/BiP and phosphorylated protein kinase RNA-like endoplasmic reticulum (ER) kinase (p-PERK). RESULTS: GRP78/BiP was increased to a greater extent in DLB and PDD patients compared with AD and control subjects in cingulate gyrus and parietal cortex. However, there were no changes in the prefrontal and temporal cortices. There was a significant positive correlation between GRP78/BiP level and α-synuclein pathology in the cingulate gyrus, while AD-type pathology showed an inverse correlation relationship in the parietal cortex. CONCLUSION: Overall, these results give emphasis to the role of UPR in Lewy body dementias, and suggest that Lewy body degeneration, in combination with AD-type pathologies, is associated with increased UPR activation to a greater extent than AD alone, possibly as a consequence of the increasing load of ER proteins. This work also highlights a novel opportunity to explore the UPR as a therapeutic target in synuclein diseases.

Expression of ER stress markers (GRP78/BiP and PERK) in patients with tongue cancer.

The glucose-regulated protein (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK) plays a crucial role in the endoplasmic reticulum (ER) stress response. GRP78/BiP is highly elevated in various human cancers. Our study is to examine the clinicopathological significance of GRP78/BiP and PERK expression in patients with tongue cancer. A total of 85 tongue cancer patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, GLUT1, Ki-67 and microvessel density (MVD) determined by CD34.GRP78/BiP and PERK were highly expressed in 47% and 35% of all patients, respectively. GRP78/BiP disclosed a significant relationship with PERK expression, lymphatic permeation, vascular invasion, glucose metabolism and cell proliferation. The expression of GRP78/BiP was significantly higher in metastatic sites than in primary sites (79% vs. 47%, p=0.003). We found that the high expression of GRP78/BiP was proven to be an independent prognostic factor for predicting poor outcome in patients with tongue cancer. In the analysis of PFS, PERK was identified as an independent predictor. The increased GRP78/BiP expression was clarified as an independent prognostic marker for predicting worse outcome. Our study suggests that the expression of GRP78/BiP as ER stress marker is important in the pathogenesis and development of tongue cancer.

Prognostic significance of GRP78/BiP expression in patients with Stage III/IV hypopharyngeal squamous cell carcinoma.

The immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein 78 (GRP78) plays an essential role in the endoplasmic reticulum (ER) stress, and GRP78/BiP is known to be highly expressed in various human neoplasms. The clinicopathological features of GRP78/BiP expression in patients with advanced hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. The aim of this study is to elucidate the prognostic significance of GRP78/BiP for HSCC.A total of 68 patients with advanced HSCC (stage III/IV) were analyzed, and tumor specimens were stained with immunohistochemistry for GRP78/BiP, Ki-67, and microvessel density (MVD), as determined through CD34 and p53 levels. GRP78/BiP was highly expressed in 80.8% (55/68) of all patients. The expression level of GRP78/BiP disclosed no significant relationship with any variables. Multivariate analysis confirmed that low expression of GRP78/BiP was an independent prognostic factor for predicting poor overall survival and progression-free survival in patients with advanced HSCC. The decreasing expression of GRP78/BiP was identified as a significant predictor related to shorter survival duration after surgery for advanced HSCC. Our study suggests that the reduced expression of GRP78/BiP contributes to worse survival for patients with advanced head and neck cancer.

Role of endoplasmic reticulum stress in rheumatoid arthritis pathogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.

Expression of GRP78 and its copartners in HEK293 and pancreatic cancer cell lines (BxPC-3/PANC-1) exposed to MRI and CT contrast agents.

Endoplasmic reticulum (ER) stress-associated chaperones trigger a defense mechanism called as unfolded protein response (UPR) which can manage apoptosis and be determinative in cell fate. Both anticancer drug effects and potential toxicity effects of magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents were aimed to be evaluated. For this purpose, we investigated expression profiles of endoplasmic reticulum stress-associated chaperone molecules in human pancreatic tumor lines BxPC-3 and PANC-1 and control human embryonic kidney cells 293 (HEK293) induced with a variety of gadolinium and iohexol contrast agents. Protein expression levels of ER stress-associated chaperones (master regulator: GRP78/Bip and its copartners: Calnexin, Ero1, PDI, CHOP, IRE1α and PERK) were evaluated with Western blotting. Expression levels at mRNA level were also assessed for GRP78/Bip and CHOP with real-time PCR. Induction of cells was carried out with four different Gd-based contrast agents (GBCAs): (Dotarem, Optimark, Primovist and Gadovist) and two different iohexol agents (Omnipol, Omnipaque). CT contrast agents tested in the study did not result in significant ER stress in HEK293 cells. However, they do not seem to have theranostic potential in pancreas cancer through ER pathway. The potential efficiency of macrocyclic MRI contrast agents to provoke apoptosis via ER stress-associated chaperones in BxPC-3 cells lends credibility for their future theranostic use in pancreas cancer as long as undesired toxicity effects were carefully considered. ER stress markers and/or contrast agents seem to have promising potential to be translated into the clinical practice to manage pancreas cancer progression.

Expression of ER stress markers (GRP78 and PERK) in experimental nephrotoxicity induced by cisplatin and gentamicin: roles of inflammatory response and oxidative stress.

This study aimed to establish the relationship between two endoplasmic reticulum (ER) stress proteins, glucose-regulated protein 78 (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK), and oxidative stress markers in cisplatin (CIS)-induced and gentamicin (GEN)-induced nephrotoxicity.The study consisted of five groups: control (saline solution only), CIS D2 (2.5 mg/kg for 2 days), CIS D7 (2.5 mg/kg for 7 days), GEN D2 (160 mg/kg for 2 days), and GEN D7 (160 mg/kg for 7 days). All rats were sacrificed 24 h after the last injection for standard clinical chemistry, and ultrastructural and histological evaluation of the kidney.CIS and GEN increased blood urea nitrogen (BUN) and serum creatinine (Cr) levels, as well as total oxidant status (TOS), while decreasing total antioxidant status (TAS) level in CIS D7 and GEN D7 groups. Histopathological and ultrastructural findings were also consistent with renal tubular damage. In addition, expression of markers of renal inflammation (tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß)) and ER stress markers (GRP78 and PERK) was significantly increased in the kidney tissue of rats treated with CIS and GEN for 7 days.These findings suggest that CIS and GEN administration for 7 days aggravates nephrotoxicity through the enhancement of oxidative stress, inflammation, and ER stress-related markers. As a result, the recommended course of action is to utilize CIS and GEN as an immediate but brief induction therapy, stopping after 3 days and switching to other drugs instead.

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells.

Introduction: Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. Methods: The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Results: Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. Discussion: These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

Ryanodine receptor type 1 content decrease-induced endoplasmic reticulum stress is a hallmark of myopathies.

BACKGROUND: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. METHODS: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RESULTS: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb-girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P < 0.001) and mitochondrial myopathy (-71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P < 0.001), Ca2+ transfer to mitochondria (-48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. CONCLUSIONS: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.

HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR).

The identification of small molecules and natural product extracts that enhance or interfere with the productivity of protein folding in the endoplasmic reticulum (ER) has the potential to improve a wide variety of human pathologies. Every protein that is destined for a lysosome, integral to the cell membrane, or secreted, is folded, post-translationally modified, and exported to the cytoplasm from the ER-Golgi complex. The following protocols have successfully employed several high-fidelity cell-based luciferase high-throughput screens (HTS) to identify activators and inhibitors of ER stress and the unfolded protein response (UPR).

Endoplasmic Reticulum Stress Is Involved in Muscular Pathogenesis in Idiopathic Inflammatory Myopathies.

Objectives: Endoplasmic reticulum (ER) stress plays pivotal roles in the regulation of skeletal muscle damage and dysfunction in multiple disease conditions. We postulate the activation of ER stress in idiopathic inflammatory myopathies (IIM). Methods: Thirty-seven patients with immune-mediated necrotizing myopathy (IMNM), 21 patients with dermatomyositis (DM), 6 patients with anti-synthetase syndrome (ASS), and 10 controls were enrolled. The expression of ER stress-induced autophagy pathway was detected using histological sections, Western blot, and real-time quantitative Polymerase Chain Reaction. Results: ER stress-induced autophagy pathway was activated in biopsied muscle of patients with IMNM, DM, and ASS. The ER chaperone protein, glucose-regulated protein 78 (GRP78)/BiP expression in skeletal muscle correlated with autophagy, myofiber atrophy, myonecrosis, myoregeneration, and disease activity in IMNM. Conclusion: ER stress was involved in patients with IIM and correlates with disease activity in IMNM. ER stress response may be responsible for skeletal muscle damage and repair in IIM.

Oxidative damage and mitochondrial functionality in hearts from KO UCP3 mice housed at thermoneutrality.

The antioxidant role of mitochondrial uncoupling protein 3 (UCP3) is controversial. This work aimed to investigate the effects of UCP3 on the heart of mice housed at thermoneutral temperature, an experimental condition that avoids the effects of thermoregulation on mitochondrial activity and redox homeostasis, preventing the alterations related to these processes from confusing the results caused by the lack of UCP3. WT and KO UCP3 mice were acclimatized at 30 °C for 4 weeks and hearts were used to evaluate metabolic capacity and redox state. Tissue and mitochondrial respiration, the activities of the mitochondrial complexes, and the protein expression of mitochondrial complexes markers furnished information on mitochondrial functionality. The levels of lipid and protein oxidative damage markers, the activity of antioxidant enzymes, the reactive oxygen species levels, and the susceptibility to in vitro Fe-ascorbate-induced oxidative stress furnished information on redox state. UCP3 ablation reduced tissue and mitochondrial respiratory capacities, not affecting the mitochondrial content. In KO UCP3 mice, the mitochondrial complexes activities were lower than in WT without changes in their content. These effects were accompanied by an increase in the level of oxidative stress markers, ROS content, and in vitro susceptibility to oxidative stress, notwithstanding that the activities of antioxidant enzymes were not affected by UCP3 ablation. Such modifications are also associated with enhanced activation/phosphorylation of EIF2α, a marker of integrated stress response and endoplasmic reticulum stress (GRP778 BIP). The lack of UCP3 makes the heart more prone to oxidative insult by reducing oxygen consumption and increasing ROS. Our results demonstrate that UCP3 helps the cell to preserve mitochondrial function by mitigating oxidative stress.

GRP78 Overexpression Triggers PINK1-IP3R-Mediated Neuroprotective Mitophagy.

An experimental model of spinal root avulsion (RA) is useful to study causal molecular programs that drive retrograde neurodegeneration after neuron-target disconnection. This neurodegenerative process shares common characteristics with neuronal disease-related processes such as the presence of endoplasmic reticulum (ER) stress and autophagy flux blockage. We previously found that the overexpression of GRP78 promoted motoneuronal neuroprotection after RA. After that, we aimed to unravel the underlying mechanism by carrying out a comparative unbiased proteomic analysis and pharmacological and genetic interventions. Unexpectedly, mitochondrial factors turned out to be most altered when GRP78 was overexpressed, and the abundance of engulfed mitochondria, a hallmark of mitophagy, was also observed by electronic microscopy in RA-injured motoneurons after GRP78 overexpression. In addition, GRP78 overexpression increased LC3-mitochondria tagging, promoted PINK1 translocation, mitophagy induction, and recovered mitochondrial function in ER-stressed cells. Lastly, we found that GRP78-promoted pro-survival mitophagy was mediated by PINK1 and IP3R in our in vitro model of motoneuronal death. This data indicates a novel relationship between the GRP78 chaperone and mitophagy, opening novel therapeutical options for drug design to achieve neuroprotection.

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum.

P-glycoprotein (P-gp, ABCB1 member of the ABC (ATP-binding cassette) transporter family) localized in leukemia cell plasma membranes is known to reduce cell sensitivity to a large but well-defined group of chemicals known as P-gp substrates. However, we found previously that P-gp-positive sublines of L1210 murine leukemia cells (R and T) but not parental P-gp-negative parental cells (S) are resistant to the endoplasmic reticulum (ER) stressor tunicamycin (an N-glycosylation inhibitor). Here, we elucidated the mechanism of tunicamycin resistance in P-gp-positive cells. We found that tunicamycin at a sublethal concentration of 0.1 µM induced retention of the cells in the G1 phase of the cell cycle only in the P-gp negative variant of L1210 cells. P-gp-positive L1210 cell variants had higher expression of the ER stress chaperone GRP78/BiP compared to that of P-gp-negative cells, in which tunicamycin induced larger upregulation of CHOP (C/EBP homologous protein). Transfection of the sensitive P-gp-negative cells with plasmids containing GRP78/BiP antagonized tunicamycin-induced CHOP expression and reduced tunicamycin-induced arrest of cells in the G1 phase of the cell cycle. Taken together, these data suggest that the resistance of P-gp-positive cells to tunicamycin is due to increased levels of GRP78/BiP, which is overexpressed in both resistant variants of L1210 cells.

MicroRNAs and Xenobiotic Toxicity: An Overview.

The advent of new technologies has paved the rise of various chemicals that are being employed in industrial as well as consumer products. This leads to the accumulation of these xenobiotic compounds in the environment where they pose a serious threat to both target and non-target species. miRNAs are one of the key epigenetic mechanisms that have been associated with toxicity by modulating the gene expression post-transcriptionally. Here, we provide a comprehensive view on miRNA biogenesis, their mechanism of action and, their possible role in xenobiotic toxicity. Further, we review the recent in vitro and in vivo studies involved in xenobiotic exposure induced miRNA alterations and the mRNA-miRNA interactions. Finally, we address the challenges associated with the miRNAs in toxicological studies.