Luteolin Protects Against 6-Hydoroxydopamine-Induced Cell Death via an Upregulation of HRD1 and SEL1L.

Parkinson's Disease (PD) is caused by many factors and endoplasmic reticulum (ER) stress is considered as one of the responsible factors for it. ER stress induces the activation of the ubiquitin-proteasome system to degrade unfolded proteins and suppress cell death. The ubiquitin ligase 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation 1 (HRD1) and its stabilizing molecule, the suppressor/enhancer lin-12-like (SEL1L), can suppress the ER stress via the ubiquitin-proteasome system, and that HRD1 can also suppress cell death in familial and nonfamilial PD models. These findings indicate that HRD1 and SEL1L might be key proteins for the treatment of PD. Our study aimed to identify the compounds with the effects of upregulating the HRD1 expression and suppressing neuronal cell death in a 6-hydroxydopamine (6-OHDA)-induced cellular PD model. Our screening by the Drug Gene Budger, a drug repositioning tool, identified luteolin as a candidate compound for the desired modulation of the HRD1 expression. Subsequently, we confirmed that low concentrations of luteolin did not show cytotoxicity in SH-SY5Y cells, and used these low concentrations in the subsequent experiments. Next, we demonsrated that luteolin increased HRD1 and SEL1L mRNA levels and protein expressions. Furthermore, luteolin inhibited 6-OHDA-induced cell death and suppressed ER stress response caused by exposure to 6-OHDA. Finally, luteolin did not reppress 6-OHDA-induced cell death when expression of HRD1 or SEL1L was suppressed by RNA interference. These findings suggest that luteolin might be a novel therapeutic agent for PD due to its ability to suppress ER stress through the activation of HRD1 and SEL1L.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Association of STAT3, CYP3A5, and ABCG2 Polymorphisms With Osimertinib-induced Adverse Events in NSCLC Patients.

BACKGROUND/AIM: Osimertinib is a key drug for treating epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). Genetic differences may be associated to adverse events (AEs) induced by osimertinib. This retrospective observational multicenter study evaluated the association of genotypes, including STAT3 -1697C>G, CYP3A5 6986A>G, and ABCG2 421C>A, with the incidence of osimertinib-induced AEs in patients with EGFR mutation-positive NSCLC. PATIENTS AND METHODS: A total of 85 patients treated with osimertinib (Institution A: 33 patients, Institution B: 52 patients) were enrolled in the study. Single nucleotide polymorphisms were determined by real-time PCR, and the incidence of AEs was compared for each genotype. RESULTS: Paronychia incidence was 59% for the CC genotype, 19% for the CG genotype, and 19% for the GG genotype at STAT3 -1697C>G. A genotype-related trend was observed (Cochran-Armitage test, p=0.009). Multivariate analysis showed that the CC genotype at STAT3 -1697C>G and female sex were significant independent factors associated with paronychia [odds ratio (OR)=6.41, 95% confidence interval (CI)=1.94-21.20 and OR=3.40, 95%CI=1.03-11.22, respectively]. The incidence of diarrhea was 53% for the CC genotype, 30% for the AC genotype, and 29% for the AA genotype at ABCG2 421C>A, and a genotype-related trend was observed (p=0.048). However, the CC genotype at ABCG2 421C>A was not a significant independent factor associated with diarrhea in multivariate analysis. No significant associations were detected between other polymorphisms and the incidence of AEs. CONCLUSION: STAT3 -1697C>G may be a novel risk factor for osimertinib-induced paronychia in patients with NSCLC.

7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

MicroRNA-101 Regulates 6-Hydroxydopamine-Induced Cell Death by Targeting Suppressor/Enhancer Lin-12-Like in SH-SY5Y Cells.

Endoplasmic reticulum (ER) stress has been reported as a cause of Parkinson's disease (PD). We have previously reported that the ubiquitin ligase HMG-CoA reductase degradation 1 (HRD1) and its stabilizing factor suppressor/enhancer lin-12-like (SEL1L) participate in the ER stress. In addition, we recently demonstrated that neuronal cell death is enhanced in the cellular PD model when SEL1L expression is suppressed compared with cell death when HRD1 expression is suppressed. This finding suggests that SEL1L is a critical key molecule in the strategy for PD therapy. Thus, investigation into whether microRNAs (miRNAs) regulate SEL1L expression in neurons should be interesting because relationships between miRNAs and the development of neurological diseases such as PD have been reported in recent years. In this study, using miRNA databases and previous reports, we searched for miRNAs that could regulate SEL1L expression and examined the effects of this regulation on cell death in PD models created by 6-hydroxydopamine (6-OHDA). Five miRNAs were identified as candidate miRNAs that could modulate SEL1L expression. Next, SH-SY5Y cells were exposed to 6-OHDA, following which miR-101 expression was found to be inversely correlated with SEL1L expression. Therefore, we selected miR-101 as a candidate miRNA for SEL1L modulation. We confirmed that miR-101 directly targets the SEL1L 3' untranslated region, and an miR-101 mimic suppressed the 6-OHDA-induced increase in SEL1L expression and enhanced cell death. Furthermore, an miR-101 inhibitor suppressed this response. These results suggest that miR-101 regulates SEL1L expression and may serve as a new target for PD therapy.

Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of Synechocystis sp. PCC 6803 metabolism.

The present study attempted a computer simulation of the metabolism of a model cyanobacteria, Synechocystis sp. PCC 6803 (PCC 6803) to predict allosteric inhibitions that are likely to occur in photoautotrophic and mixotrophic conditions as well as in a metabolically engineered strain. PCC 6803 is a promising host for direct biochemical production from CO2; however, further investigation of allosteric regulation is required for rational metabolic engineering to produce target compounds. Herein, ensemble modeling of microbial metabolism was applied to build accurate predictive models by synthesizing the results of multiple models with different parameter sets into a single score to identify plausible allosteric inhibitions. The data driven-computer simulation using metabolic flux, enzyme abundance, and metabolite concentration data successfully identified candidates for allosteric inhibition. The enzyme assay experiment using the recombinant protein confirmed isocitrate was a non-competitive inhibitor of phosphoribulokinase as a novel allosteric regulation of cyanobacteria metabolism.

Time-resolved analysis of short term metabolic adaptation at dark transition in Synechocystis sp. PCC 6803.

In photosynthetic organisms, such as cyanobacteria, ATP and NADPH are generated through the light reaction, and then are used for CO2 fixation in the dark reaction. As light intensity always fluctuates under natural conditions, balancing the cofactor regeneration and consumption is essential to maintain active CO2 fixation as well as for metabolic engineering of strains that produce biochemicals. In this study, a time-resolved metabolome analysis of Synechocystis sp. PCC 6803 (PCC6803) was conducted to investigate a metabolic adaptation at 0-15 min after a sudden shift from light to dark conditions. Rapid accumulation of sedoheptulose 7-phosphate, ribulose 5-phosphate, xylulose 5-phosphate, and 6-phosphogluconate suggested that the central metabolism of PCC6803 was regulated by inactivation of phosphoribulokinase and activation of glucose-6-phosphate dehydrogenase (G6PDH) probably via the redox regulation. The culture and metabolic profile of the Δzwf strain lacking G6PDH showed that the role of G6PDH in regeneration of NADPH could be complemented by the activation of isocitrate dehydrogenase in the TCA cycle, indicating the importance of the rapid regulation of NADPH regeneration after the shift to dark conditions. The mechanism underlying metabolic regulation is also useful for metabolic engineering of PCC6803, as the Δzwf strain produced higher amount of organic acids than wild type.

Transomics data-driven, ensemble kinetic modeling for system-level understanding and engineering of the cyanobacteria central metabolism.

In silico kinetic modeling is an essential tool for rationally designing metabolically engineered organisms based on a system-level understanding of their regulatory mechanisms. However, an estimation of enzyme parameters has been a bottleneck in the computer simulation of metabolic dynamics. In this study, the ensemble-modeling approach was integrated with the transomics data to construct kinetic models. Kinetic metabolic models of a photosynthetic bacterium, Synechocystis sp. PCC 6803, were constructed to identify engineering targets for improving ethanol production based on an understanding of metabolic regulatory systems. A kinetic model ensemble was constructed by randomly sampling parameters, and the best 100 models were selected by comparing predicted metabolic state with a measured dataset, including metabolic flux, metabolite concentrations, and protein abundance data. Metabolic control analysis using the model ensemble revealed that a large pool size of 3-phosphoglycerate could be a metabolic buffer responsible for the stability of the Calvin-Benson cycle, and also identified that phosphoglycerate kinase (PGK) is a promising engineering target to improve a pyruvate supply such as for ethanol production. Overexpression of PGK in the metabolically engineered PCC 6803 strain showed that the specific ethanol production rate and ethanol titers at 48 h were 1.23- and 1.37-fold greater than that of the control strain. PGK is useful for future metabolic engineering since pyruvate is a common precursor for the biosynthesis of various chemicals.