Molecular characterization of the ER stress-inducible factor CRELD2.

Previously, we found by constructing various luciferase reporters that a well-conserved ATF6-binding element in the CRELD2 promoter is activated by transient ATF6 overexpression. In this study, we established ATF6-deficient and ATF4-deficient cell lines to analyze CRELD2 mRNA and protein expression together with that of other ER stress-inducible factors. Our results showed that ATF6 deficiency markedly suppressed tunicamycin (Tm)-induced expression of unglycosylated CRELD2. This reduction reflected a decrease in the CRELD2 transcription level. On the other hand, a putative ATF4-binding site in the mouse CRELD2 promoter did not respond to Tm stimulation, but ATF4 loss resulted in reductions in CRELD2 mRNA and protein expression, accompanied by a decrease in Tm-induced ATF6 expression. In contrast, transient suppression of GADD34, an ATF4 downstream factor, suppressed Tm-induced CRELD2 protein expression without a decrease in ATF6 protein expression. Furthermore, we investigated the association of CRELD2 with a well-known ERAD substrate, namely, an α1-antitripsin truncation mutant, NHK, by generating various CRELD2 and NHK constructs. Coimmunoprecipitation of these proteins was observed only when the cysteine in the CXXC motif on the N-terminal side of CRELD2 was replaced with alanine, and the interaction between the two was found to be disulfide bond-independent. Taken together, these findings indicate that CRELD2 expression is regulated by multiple factors via transcriptional and posttranscriptional mechanisms. In addition, the N-terminal structure of CRELD2, including the CXXC motif, was suggested to play a role in the association of the target proteins. In the future, the identification and characterization of factors interacting with CRELD2 will be useful for understanding protein homeostasis under various ER stress conditions.

Melanosomal localization is required for GIF-2115/2250 to inhibit melanogenesis in B16F10 melanoma cells.

OBJECTIVE: Tyrosinase inhibitors suppress melanogenesis in melanocytes. During a screening for tyrosinase inhibitors, however, we noticed some discrepancies in inhibitory efficacies between melanocytes and in vitro assays. The compound (S)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-indan-1-amine (GIF-2115) exerts antioxidative stress activity upon accumulation in late endosomes and lysosomes. GIF-2115 was also identified as a potent antimelanogenic reagent in B16F10 mouse melanoma cells. GIF-2115 inhibited the activity of mushroom tyrosinase and the lysates of B16F10 cells. However, structure-activity relationship studies indicated that GIF-2238, which lacks the benzene ring in the aminoindan structure of GIF-2115, inhibited tyrosinase activity in vitro but did not inhibit melanogenesis in B16F10 cells. The aim of the present study is to show the importance of the intracellular distribution of tyrosinase inhibitors in exerting their antimelanogenic activity in melanocytes. METHODS: The intracellular distribution of compounds was monitored by linking with the fluorescent group of 7-nitro-2,1,3-benzoxadiazole (NBD). To mislocalize GIF-2115 to mitochondria, the mitochondria-preferring fluoroprobe ATTO565 was used. RESULTS: We reconfirmed the localization of GIF-2250 (GIF-2115-NBD) not only to matured but also to early-stage melanosomes. Although GIF-2286 (GIF-2238-NBD) maintained tyrosinase inhibitory activity, it did not show specific intracellular localization. Moreover, when GIF-2115 was linked with ATTO565, the resultant compound GIF-2265 did not inhibit melanogenesis in B16F10 cells, despite its strong tyrosinase inhibitory activity. CONCLUSION: These results suggest that melanosomal localization is essential for the antimelanogenic activity of GIF-2115, and GIF-2115 derivatives may be new guides for drugs to endosomes and lysosomes as well as melanosomes.

OBJECTIF: Les inhibiteurs de la tyrosinase suppriment la mélanogenèse dans les mélanocytes. Lors d'un criblage d'inhibiteurs de la tyrosinase, cependant, nous avons remarqué des différences dans les efficacités inhibitrices entre les mélanocytes et les essais in vitro. Le composé (S)-N-{3-[4-(diméthylamino)phényl]propyl}-N-méthyl-indan-1-amine (GIF-2115) exerce une activité antioxydante en cas de stress lors de l'accumulation dans les endosomes tardifs et les lysosomes. GIF-2115 a également été identifié comme un puissant réactif antimélanogène dans les cellules de mélanome murin B16F10. GIF-2115 a inhibé l'activité de la tyrosinase de champignon et les lysats des cellules B16F10. Cependant, des études de relation structure-activité ont indiqué que GIF-2238, à qui il manque l'anneau benzénique dans la structure aminoindan de GIF-2115, inhibait l'activité de la tyrosinase in vitro mais n'inhibait pas la mélanogenèse dans les cellules B16F10. L'objectif de la présente étude est de montrer l'importance de la distribution intracellulaire des inhibiteurs de la tyrosinase dans l'exercice de leur activité antimélanogène dans les mélanocytes. MÉTHODES: La distribution intracellulaire des composés a été surveillée en les liant au groupe fluorescent de la 7-nitro-2,1,3-benzoxadiazole (NBD). Pour délocaliser GIF-2115 vers les mitochondries, le fluorophore ATTO565 préférant les mitochondries a été utilisé. RÉSULTATS: Nous avons confirmé la localisation de GIF-2250 (GIF-2115-NBD) non seulement dans les mélanosomes matures mais aussi dans les mélanosomes à un stade précoce. Bien que GIF-2286 (GIF-2238-NBD) ait maintenu une activité inhibitrice de la tyrosinase, il n'a pas montré de localisation intracellulaire spécifique. De plus, lorsque GIF-2115 a été lié à ATTO565, le composé résultant GIF-2265 n'a pas inhibé la mélanogenèse dans les cellules B16F10, malgré son activité inhibitrice de la tyrosinase forte. CONCLUSION: Ces résultats suggèrent que la localisation dans les mélanosomes est essentielle pour l'activité antimélanogène de GIF-2115, et que les dérivés de GIF-2115 peuvent être de nouveaux guides pour les médicaments vers les endosomes et les lysosomes ainsi que les mélanosomes.

Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.

BACKGROUND: Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes. METHODS AND RESULTS: Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy. CONCLUSIONS: In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.

Antiferroptotic Activities of Oxindole GIF-0726-r Derivatives: Involvement of Ferrous Iron Coordination and Free-Radical Scavenging Capacities.

Ferroptosis and oxytosis are iron- and oxidative stress-dependent cell death pathways strongly implicated in neurodegenerative diseases, cancers, and metabolic disorders. Therefore, specific inhibitors may have broad clinical applications. We previously reported that 3-[4-(dimethylamino)benzyl]-2-oxindole (GIF-0726-r) and derivatives protected the mouse hippocampal cell line HT22 against oxytosis/ferroptosis by suppressing reactive oxygen species (ROS) accumulation. In this study, we evaluated the biological activities of GIF-0726-r derivatives with modifications at the oxindole skeleton and other positions. The addition of a methyl, nitro, or bromo group to C-5 of the oxindole skeleton enhanced antiferroptotic efficacy on HT22 cells during membrane cystine-glutamate antiporter inhibition and ensued intracellular glutathione depletion. In contrast, the substitution of the dimethylamino group on the side chain phenyl ring with a methyl, nitro, or amine group dramatically suppressed antiferroptotic activity regardless of other modifications. Compounds with antiferroptotic activity also directly scavenged ROS and decreased free ferrous ions in both HT22 cells and cell-free reactions while those compounds without antiferroptotic activity had little effect on either ROS or ferrous-ion concentration. Unlike oxindole compounds, which we have previously reported, the antiferroptotic compounds had little effect on the nuclear factor erythroid-2-related factor 2-antioxidant response element pathway. Oxindole GIF-0726-r derivatives with a 4-(dimethylamino)benzyl moiety at C-3 and some types of bulky group at C-5 (whether electron-donating or electron-withdrawing) can suppress ferroptosis, warranting safety and efficacy evaluations in animal models of disease.

Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells.

OSW-1, a steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, is a promising compound for an anticancer drug; however, its cytotoxic mechanisms have not been fully elucidated. Therefore, we analyzed the stress responses triggered by OSW-1 in the mouse neuroblastoma cell line Neuro2a by comparing it with brefeldin A (BFA), a Golgi apparatus-disrupting reagent. Among the Golgi stress sensors TFE3/TFEB and CREB3, OSW-1 induced dephosphorylation of TFE3/TFEB but not cleavage of CREB3, and induction of the ER stress-inducible genes GADD153 and GADD34 was slight. On the other hand, the induction of LC3-II, an autophagy marker, was more pronounced than the BFA stimulation. To elucidate OSW-1-induced gene expression, we performed a comprehensive gene analysis using a microarray method and observed changes in numerous genes involved in lipid metabolism, such as cholesterol, and in the regulation of the ER-Golgi apparatus. Abnormalities in ER-Golgi transport were also evident in the examination of secretory activity using NanoLuc-tag genes. Finally, we established Neuro2a cells lacking oxysterol-binding protein (OSBP), which were severely reduced by OSW-1, but found OSBP deficiency had little effect on OSW-1-induced cell death and the LC3-II/LC3-I ratio in Neuro2a cells. Future work to elucidate the relationship between OSW-1-induced atypical Golgi stress responses and autophagy induction may lead to the development of new anticancer agents.

Haloperidol Prevents Oxytosis/Ferroptosis by Targeting Lysosomal Ferrous Ions in a Manner Independent of Dopamine D2 and Sigma-1 Receptors.

Haloperidol is a widely used antipsychotic agent that exerts antipsychotic effects through a strong antagonism of dopamine D2 receptors. In addition, haloperidol is classified as a sigma-1 receptor (S1R) antagonist that prevents endogenous oxidative stress in cultured cells. However, pharmacological activities of haloperidol against oxidative stress remain unclear. Oxytosis/ferroptosis are iron-dependent nonapoptotic oxidative cell deaths that are regarded as two names for the same cell death pathway and the potential physiological relevance of oxytosis/ferroptosis in multiple diseases is suggested. In the present study, the effects of haloperidol on oxytosis/ferroptosis were investigated in S1R-knockdown mouse hippocampal HT22 cells. The results indicate that haloperidol is a strong inhibitor of oxytosis/ferroptosis independent of S1R. Imaging of HT22 cells with a newly developed fluorescent probe showed that haloperidol was localized to late endosomes and lysosomes and reduced the accumulation of lysosomal ferrous ions, resulting in reduced production of intracellular reactive oxygen species and inhibition of cell death. These results indicate that haloperidol is useful not only as an antipsychotic agent but also as a neuroprotective agent against endogenous oxidative stress via distinct mechanisms. Furthermore, lysosome-targeting ferroptosis inhibitors could be useful for the treatment of various diseases, including cancers, ischemia-reperfusion injury, and neurodegenerative disorders, which have been associated with ferroptosis.

Artepillin C, a major component of Brazilian green propolis, inhibits endoplasmic reticulum stress and protein aggregation.

Propolis, a compound produced by honeybees, has long been used in food and beverages to improve health and prevent diseases. We previously reported that the ethanol extracts of Brazilian green propolis and its constituents artepillin C, kaempferide, and kaempferol mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. Here, we investigated the potential of Brazilian green propolis and its constituents to protect against endoplasmic reticulum stress in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis, artepillin C, and kaempferol attenuated tunicamycin-induced unfolded protein response and cell death. Interestingly, artepillin C inhibited both tunicamycin-induced protein aggregation in HT22 cells and the spontaneous protein aggregation of mutant canine superoxide dismutase 1 (E40K-SOD1-EGFP) in Neuro2a cells. These findings indicate that in addition to oxidative stress, the ethanol extracts of Brazilian green propolis help prevent endoplasmic reticulum stress-related neuronal cell death, which is proposedly involved in several neurodegenerative diseases. Moreover, artepillin C, a major constituent of Brazilian green propolis, may exhibit chemical chaperone-like properties.

Molecular characterization of mouse CREB3 regulatory factor in Neuro2a cells.

We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length (FL) CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged FL CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.

The new live imagers MitoMM1/2 for mitochondrial visualization.

Mitochondria are eukaryotic organelles that consist of outer and inner bilayer membranes with a positive potential (H+) in the intermembrane space. This organelle plays an important role in ATP production and apoptosis. To observe the mitochondria in living cells, several fluorescent dyes (such as MitoTracker® [a standard mitochondrial imager] or rhodamine 123) have been developed. However, these reagents are unstable and exhibit a wide range of emission spectra, thereby hampering double staining results. Using recombinant DNA techniques, green or red fluorescent protein (GFP or RFP)-tagged proteins are now available for multi-color labeling of mitochondria. Here, we have discussed the development of the novel mitochondrial live imagers MitoMM1/2, derivatives of ATTO565; furthermore, MitoMM1/2 are sensitive to the membrane potential, resistant to detergents, and the fluorescence of MitoMM1/2 does not overlap with green fluorescence.

Oxindole-curcumin hybrid compound enhances the transcription of γ-glutamylcysteine ligase.

Glutathione (GSH), which is particularly important for antioxidant defenses, is synthesized in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine ligase (GCL) and GSH synthase. GCL comprises catalytic (GCLC) and regulatory subunits and catalyzes the rate-limiting step in de novo GSH synthesis. Accumulating evidence suggests that substances that stimulate GSH synthesis are therapeutic modalities for neurodegenerative disorders and schizophrenia, in which a deficit in brain GSH content has been observed. In the present study, we attempted to develop small organic compounds that increase GCLC transcription. Using HT22 cells stably expressing a luciferase reporter that contains rat GCLC promoter region (-1764 to +2), we assessed the effects of the novel neuroprotective compound oxindole and related compounds on GCLC promoter activity. Among approximately 220 synthesized compounds, five compounds increased GCLC promoter activity by >200% at a concentration of 50 µM, and 16 compounds increased promoter activity by approximately 150%. The most effective compound oxindole-curcumin hybrid GIF-2165X-G1 increased GCLC mRNA levels in HT22 mouse hippocampal cells, PC12 rat pheochromocytoma cells, and C6 rat glioma cells. Although GIF-2165X-G1 potently induced antioxidant response element (ARE)-driven transcription, the compound increased GCLC transcriptional activity through Sp1 pathway in a Keap1-Nrf2-ARE-independent manner. These results suggest that GIF-2165X-G1 itself and further modification of the compound are useful interventions for promoting neuronal survival by augmenting resistance to oxidative stress.

Transcriptional regulation of the ER stress-inducible gene Sec16B in Neuro2a cells.

Endoplasmic reticulum (ER) stress responses have been demonstrated to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we identified Sec16B as an ER stress-inducible gene by microarray analysis of brefeldin A (BFA)-inducible genes in a mouse neuroblastoma cell-line, Neuro2a. Sec16B mRNA was induced by treatment with the ER stress-inducing reagents thapsigargin (Tg) and brefeldin A in a time-dependent manner. In the genomic sequence of the mouse Sec16B gene, we found an unfolded protein response element (UPRE), which is well conserved between humans and mice. Using luciferase reporter analyses, we showed that the UPRE in the mouse Sec16B gene was functional and responded well to ER stress-inducing stimuli and spliced XBP1 (sXBP1)-overexpression. In addition, a unique ATF4-responsive sequence within the first intron of the mouse Sec16B gene was characterized. Our study may help to elucidate the regulation of trafficking through the ER-Golgi apparatus and the biogenesis of ER-derived intracellular organelles.

Repurposing of FDA-approved antivirals, antibiotics, anthelmintics, antioxidants, and cell protectives against SARS-CoV-2 papain-like protease.

SARS-CoV-2 or Coronavirus disease 19 (COVID-19) is a rapidly spreading, highly contagious, and sometimes fatal disease for which drug discovery and vaccine development are critical. SARS-CoV-2 papain-like protease (PLpro) was used to virtually screen 1697 clinical FDA-approved drugs. Among the top results expected to bind with SARS-CoV-2 PLpro strongly were three cell protectives and antioxidants (NAD+, quercitrin, and oxiglutatione), three antivirals (ritonavir, moroxydine, and zanamivir), two antimicrobials (doripenem and sulfaguanidine), two anticancer drugs, three benzimidazole anthelmintics, one antacid (famotidine), three anti-hypertensive ACE receptor blockers (candesartan, losartan, and valsartan) and other miscellaneous systemically or topically acting drugs. The binding patterns of these drugs were superior to the previously identified SARS CoV PLpro inhibitor, 6-mercaptopurine (6-MP), suggesting a potential for repurposing these drugs to treat COVID-19. The objective of drug repurposing is the rapid relocation of safe and approved drugs by bypassing the lengthy pharmacokinetic, toxicity, and preclinical phases. The ten drugs with the highest estimated docking scores with favorable pharmacokinetics were subjected to molecular dynamics (MD) simulations followed by molecular mechanics/generalized Born surface area (MM/GBSA) binding energy calculations. Phenformin, quercetin, and ritonavir all demonstrated prospective binding affinities for COVID-19 PLpro over 50 ns MD simulations, with binding energy values of -56.6, -40.9, and -37.6 kcal/mol, respectively. Energetic and structural analyses showed phenformin was more stable than quercetin and ritonavir. The list of the drugs provided herein constitutes a primer for clinical application in COVID-19 patients and guidance for further antiviral studies.Communicated by Ramaswamy H. Sarma.

Activation of protein kinase R in the manganese-induced apoptosis of PC12 cells.

Manganese neurotoxicity leads to Parkinson-like symptoms associated with the apoptotic cell death of dopaminergic neurons. Protein kinase R (PKR) is a serine/threonine-specific protein kinase that has been implicated in several cellular signal transduction pathways, including the induction of apoptosis. Here, we investigated the role of PKR in the manganese-induced apoptosis of dopamine-producing pheochromocytoma PC12 cells. Manganese (0.5 mM) induced the proteolytic cleavage of PKR and caspase-3, DNA fragmentation, and cell death, which were prevented by the co-treatment of PC12 cells with a PKR specific inhibitor, C16 in a concentration-dependent manner. C16 did not affect the manganese-induced activation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) pathway, indicating that PKR functions downstream of JNK and p38 MAPK. In contrast, C16 triggered the activation of the p44/42 MAPK (ERK1/2) pathway and induced hemoxygenase-1, both in the absence and presence of manganese. PKR is reportedly involved in endoplasmic reticulum (ER) stress-induced apoptosis. Manganese activated all three branches of the unfolded protein response in PC12 cells; however, this effect was very weak compared with the ER stress induced by the well-known ER stress inducers thapsigargin and tunicamycin. Moreover, C16 did not affect manganese-induced ER stress at concentrations that almost prevented caspase-3 activation and DNA fragmentation. These results suggest that PKR is involved in manganese-induced apoptotic cell death and stress response, such as the activation of the p44/42 MAPK pathway and the induction of hemoxygenase-1. Although manganese induced a faint, but typical, ER stress, these events contributed little to manganese-induced apoptosis.

Reduced Sulfation Enhanced Oxytosis and Ferroptosis in Mouse Hippocampal HT22 Cells.

Sulfation is a common modification of extracellular glycans, tyrosine residues on proteins, and steroid hormones, and is important in a wide variety of signaling pathways. We investigated the role of sulfation on endogenous oxidative stress, such as glutamate-induced oxytosis and erastin-induced ferroptosis, using mouse hippocampal HT22 cells. Sodium chlorate competitively inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, the high energy sulfate donor in cellular sulfation reactions. The treatment of HT22 cells with sodium chlorate decreased sulfation of heparan sulfate proteoglycans and chondroitin sulfate proteoglycans. Sodium chlorate and ß-d-xyloside, which prevents proteoglycan glycosaminoglycan chain attachment, exacerbated both glutamate- and erastin-induced cell death, suggesting that extracellular matrix influenced oxytosis and ferroptosis. Moreover, sodium chlorate enhanced the generation of reactive oxygen species and influx of extracellular Ca2+ in the process of oxytosis and ferroptosis. Interestingly, sodium chlorate did not affect antioxidant glutathione levels. Western blot analysis revealed that sodium chlorate enhanced erastin-induced c-Jun N-terminal kinase phosphorylation, which is preferentially activated by cell stress-inducing signals. Collectively, our findings indicate that sulfation is an important modification for neuroprotection against oxytosis and ferroptosis in neuronal hippocampal cells.

Novel Oxindole-Curcumin Hybrid Compound for Antioxidative Stress and Neuroprotection.

Oxidative stress plays an important role in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. The oxindole compound GIF-2165X-G1 is a hybrid molecule composed of the oxindole skeleton of the neuroprotective compound GIF-0726-r and the polyphenolic skeleton of the antioxidant curcumin. We previously reported that novel oxindole derivatives such as GIF-0726-r and GIF-2165X-G1 prevent endogenous oxidative stress-induced cell death in mouse hippocampal HT22 cells. In this study, we present a detailed investigation of the effect of GIF-2165X-G1 on endogenous oxidative stress in HT22 cells in comparison with GIF-0726-r and curcumin. GIF-2165X-G1 exhibited more potent neuroprotective activity than GIF-0726-r or curcumin and had less cytotoxicity than that observed with curcumin. Both GIF-0726-r and GIF-2165X-G1 were found to have ferrous ion chelating activity similar to that exhibited by curcumin. GIF-2165 X-G1 and curcumin induced comparable antioxidant response element transcriptional activity. Although the induction of heme oxygenase-1, an antioxidant response element-regulated gene product, was much stronger in curcumin-treated cells than in GIF-2165X-G1-treated cells, it turned out that the induction of heme oxygenase-1 is dispensable for neuroprotection. These results demonstrate that the introduction of the polyphenol skeleton of curcumin to the oxindole GIF-0726-r improves neuroprotective features. Furthermore, intrastriatal injection of GIF-2165X-G1 alleviated apomorphine-induced rotation and prevented dopaminergic neuronal loss in a 6-hydroxydopamine mouse model of Parkinson's diseases. Collectively, our novel findings indicate that the novel oxindole compound GIF-2165X-G1 serves to delay the progression of Parkinson's disease by suppressing oxidative stress.

Regulation of the ER-bound transcription factor Luman/CREB3 in HEK293 cells.

CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing approaches. Full-length CREB3 detected under resting conditions disappeared following treatment with tunicamycin, brefeldin A and nigericin. Treatment with cycloheximide and MG132 indicated that endogenous CREB3 is a proteasome substrate. Using cells deficient for the ER-associated protein degradation (ERAD) factors SEL1L and Herp, we demonstrate that SEL1L, but not Herp, plays a crucial role in the posttranslational regulation of full-length CREB3 expression. In addition, kifunensine, an α-mannosidase inhibitor, remarkably increased full-length CREB3 expression. Our study suggests that endogenous full-length CREB3 is a novel substrate for ERAD and identifies unique cellular signals distinct from those in canonical ER stress.

Effects of 2-(2-Chlorophenyl)ethylbiguanide on ERAD Component Expression in HT-29 Cells Under a Serum- and Glucose-Deprived Condition.

We recently characterized the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition and found that 2-Cl-Phen attenuated ATF4 and GRP78, typical downstream targets of the unfolded protein response (UPR), together with c-Myc protein expression in a transcriptional and posttranscriptional manner. In the current study, we focused on the expression of ER-associated protein degradation (ERAD) components after treatment with 2-Cl-Phen under a serum- and glucose-deprived condition. Among nine ER-localizing factors regulating protein quality control within the ER, the amounts of Herp, GRP78, GRP94, and OS9 proteins were significantly downregulated by treatment with 2-Cl-Phen. In particular, replacement of the culture medium with the serum- and glucose-deprived medium induced the expression of Herp protein at the early phase. This increase in Herp protein was accompanied by an increase in its mRNA, and its induction was significantly dampened by 2-Cl-Phen. However, cotreatment with a proteasome inhibitor, MG132, restored Herp expression only to a limited extent. Taken together, these results show that 2-Cl-Phen changed the expression of several ERAD components, especially by transcriptional inhibition of Herp induction by 2-Cl-Phen when it occurred at an early phase, and this finding provides new insights into understanding the mechanisms of 2-Cl-Phen-mediated cytotoxicity.

Expression analysis and functional characterization of the mouse cysteine-rich with EGF-like domains 2.

We have previously identified a novel endoplasmic reticulum (ER) stress-inducible protein, namely, cysteine-rich with EGF-like domains 2 (CRELD2), which is predominantly regulated by ATF6. However, few studies on intrinsic CRELD2 have been published. In the present study, we elucidated the expression of intrinsic CRELD2 in mouse tissues and ER stress- treated Neuro2a cells. Among nine tissues we tested, CRELD2 protein in the heart and skeletal muscles was negligible. CRELD2 expression in Neuro2a cells was induced at the late phase after treatment with tunicamycin (Tm) compared with rapid induction of growth arrest and DNA damage inducible gene 153 (GADD153). On the other hand, another ER stress inducer, thapsigargin, increased the intrinsic CRELD2 secretion from Neuro2a cells. We furthermore established CRELD2-deficient Neuro2a cells to evaluate their features. In combination with the NanoLuc complementary reporter system, which was designed to detect protein-protein interaction in living cells, CRELD2 interacted with not only CRELD2 itself but also with ER localizing proteins in Neuro2a cells. Finally, we investigated the responsiveness of CRELD2-deficient cells against Tm-treatment and found that CRELD2 deficiency did not affect the expression of genes triggered by three canonical ER stress sensors but rendered Neuro2a cells vulnerable to Tm-stimulation. Taken together, these findings provide the novel molecular features of CRELD2, and its further characterization would give new insights into understanding the ER homeostasis and ER stress-induced cellular dysfunctions.

Application of NanoBiT for Monitoring Dimerization of the Null Hong Kong Variant of α-1-Antitrypsin, NHK, in Living Cells.

In this study, we investigated expression and dimerization of an ER-associated degradation (ERAD) substrate, a null Hong Kong variant of α-1-antitrypsin (NHK) using immunoblotting assay and a novel NanoLuc complementary reporter system called the NanoBiT (NB) assay. This NB-tagged NHK made it possible to monitor the intra- and extracellular status of NHK in living cells. The values for this NB assay fluctuated in response to distinct pharmacological stimuli and co-transfection of several ERAD-related factors. We then focused on mesencephalic astrocyte-derived neurotrophic factor (MANF), an unclarified ATF6/IRE1-downstream target, and established MANF-deficient Neuro2a (N2a) cells using CRISPR/Cas9 system. MANF-deficient N2a significantly elevated OS-9 protein after tunicamycin treatment; however, no specific differences in intra- and extracellular status of NHK protein were observed between wild-type and MANF-deficient cells. Taken together, intrinsic MANF in N2a cells is not strongly associated with the accumulation and clearance of unfolded proteins within the ER under current condition, but this novel NB assay is a useful approach for characterizing the protein status including ERAD substrates.

Elucidating the rapid action of 2-(2-chlorophenyl)ethylbiguanide on HT-29 cells under a serum- and glucose-deprived condition.

We recently demonstrated the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition. In that study, we showed that the ATF6 arm of the ER stress pathway and c-Myc expression were downregulated 12 h after the treatment with 2-Cl-Phen. Through characterization of intracellular events at the early phase of the 2-Cl-Phen treatment before noticeable morphological changes, we found rapid fluctuations in the c-Myc and ATF4 proteins but not in their mRNAs in 2-Cl-Phen-treated HT-29 cells under the serum- and glucose-deprived condition. The 2-Cl-Phen-mediated downregulation of ATF4 protein was not paralleled by the phosphorylation status of PERK and eIF2α. Reduction of c-Myc expression by 2-Cl-Phen was more profound than that of ATF4 expression, and phosphorylated c-Myc was downregulated within 2 h. Pharmacological studies on the expression of c-Myc and ATF4 proteins showed that this decrease was mediated through proteasomal degradation but not by autophagy. Interestingly, treatment with lithium chloride, which is a well-known inhibitor of GSK3ß, partially recovered the expression of ATF4 protein, but its effect on the level of total c-Myc protein was negligible. Treatment with 2-Cl-Phen increased the expression of phosphorylated AMPK, but Compound C, an AMPK inhibitor, did not influence the expression of c-Myc protein in HT-29 cells. Finally, we observed that 2-Cl-Phen partially attenuated the gene expression of integrin subunit α1 (ITGA1), a downstream target of c-Myc. Taken together, these results show that 2-Cl-Phen rapidly downregulated the expression of c-Myc in addition to ER stress responses in a post-translational manner. Further elucidation and improvement of this multi-target-directed compound will provide new insights for developing therapeutic strategies against cancer.