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The predicted molecular pathway of SP1 involved in the inhibition of autophagy-mediated apoptosis induced by starvation. SP1 suppresses the EBSS-induced increase in ATG7 level, and regulates the basal Beclin-1 level.
Source publication
Autophagy is a natural physiological process, and it induces the lysosomal degradation of intracellular components in response to environmental stresses, including nutrient starvation. Although an adequate autophagy level helps in cell survival, excessive autophagy triggered by stress such as starvation leads to autophagy-mediated apoptosis. Chines...
Contexts in source publication
Context 1
... LC3-I conversion to LC3-II is regulated by the ATG system, including the ATG7 level during starvation. As ATG7 expression level was significantly suppressed by SP1 expression in cells (Figure 4), this explains how the LC3-II formation followed by apoptosis induction was suppressed by SP1 (Figure 7). Moreover, the basal Beclin-1 level was low in the SP1-expressing cells and reduced further after the EBSS treatment. ...
Context 2
... finding suggests that decreased Beclin-1 level in the SP1-expressing cells reduces mitochondrial damage, and in turn, ROS generation. Because increased ROS levels trigger apoptosis cascades, reduction of ROS production by SP1 under starvation may suppress cell death (Figure 7). Thus, these dual effects of SP1 (ATG7 suppression, low Beclin-1 basal level) on autophagy synergistically alleviate intracellular stress, increase the resistance of cells to starvation, and suppress autophagy-mediated apoptosis. ...
Citations
... For increasing viabitlity of CHO during culture most manipulations have been performed on genes involved in apoptosis and autophagy pathways. Examples include increasing the expression of anti-apoptotic proteins such as BCL-2 [9], BCL-XL, MCL1 [10] and reducing the expression of apoptotic-inducing proteins such as BAK, BAX [11], caspases 3, 9,8 [12, 13] and autophagy pathway related genes [14]. Although, these strategies have led to increased cell survival and production yield, targeting the genes that only regulate one apoptosis pathway could lead to activation of other signaling pathways related to cell death [15]. ...
Background
Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms.
Methods and results
In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively.
Conclusions
This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.
... Knockout of the silkworm SP 1 via the CRISPR/Cas 9 system resulted in a significant reduction of the hatching rate of silkworm eggs, accompanied by reduced vitellogenin mRNA expression (Zhu et al., 2019). In addition, it has been reported that SPs can inhibit cell apoptosis, melanin formation and active antioxidant peptide formation (Kang and Rhee, 2019;Lee et al., 2015;Levine et al., 1996;Li et al., 2020;Rhee et al., 2007;Yu et al., 2013). ...
Insect serum proteins, also termed storage proteins (SPs), are hexamer proteins that form amino acid reservoirs important for the development of pupae and embryos in most insects. In this study, we investigated the SP genes expression and regulation pathways in silkworms (Bombyx mori). We observed that B. mori SPs (BmSPs) in the fat body of larvae were strongly decreased by starvation, suggesting they respond to nutrition deprivation. Further, we examined the relationship between BmSP expression and the insulin-like signaling pathway (ILS) to study the regulation of BmSPs expression. The results showed that insulin up-regulated the expression of BmSPs, but an inhibitor of the ILS pathway protein PI3K downregulated the expression of BmSPs in B. mori larvae. Similar results were observed in cultured fat body in vitro and BmE cells. We then over-expressed FoxO, an ILS transcriptional factor, in BmE cells and B. mori larvae to further verify the regulatory role of ILS on expression of BmSPs and found BmFoxO negatively regulates the expression of BmSPs in both BmE cells and larvae. Moreover, BmFoxO was dephosphorylated and translocated from the cytoplasm to the nucleus under starvation treatment. Finally, an element on −2627–2644 bp upstream of the transcription start site of BmSP1 was identified as the binding site of BmFoxO by electrophoretic mobility shift assay and verified by chromatin immunoprecipitation. In summary, our results indicate that nutrient uptake triggers the expression of BmSPs via the ILS/FoxO signaling pathway. This study provides a reference for further study on the expression and regulation of insect SP genes.
... SP1 is one of SPs with high methionine content. It was used as amino acid reservoir [38] and has many functions including antioxidant activity [44] and inhibiting cell apoptosis [45]. In addition, SP1 can bind to LP6 protein to play an important regulatory role in the growth and development and innate immunity in silkworm [46]. ...
Acetylation is a highly conservative and reversible post-translational modification. Acetylation modification can regulate gene expression by altering protein function and is widely identified in an increasing number of species. Previously, the acetylated proteome of silkworm was identified by combining acetylated polypeptide enrichment with nano-HPLC/MS/MS; the identification revealed that the SP proteins (SPs) were high acetylated. In this study, the acetylation of SP1, one of the SPs, was further confirmed using immunoprecipitation (IP) and Western blotting. Then, we found the acetylation could upregulate SP1 protein expression by enhancing the protein stability. Further research found that the acetylation of SP1 protein can competitively inhibit its ubiquitination and thus improve the stability and cell accumulation of SP1 protein by inhibiting the ubiquitin-mediated proteasome degradation pathway. This result provides a basis for acetylation to regulate the nutrient storage and utilization of silkworm.
... Apoptotic Henrietta Lacks (HeLa) cells cultured in the medium supplemented with silkworm hemolymph able to maintain cell death rate at 25% during the culture period whereas cell death rate was increased from 39.5% at 48 h to 45.5% at 60 h in medium without hemolymph [197]. In HeLa cells, both intracellular expression and extracellular supplementation of silkworm storage protein 1 and its domain 1 (SPD1) successfully inhibited apoptosis and autophagy-mediated apoptosis [198][199][200]. In addition, adding cell derived exosomes [201] to CHO cell culture medium enhances the resistance to apoptosis during the culturing process. ...
Mammalian cell cultures are widely used in the biopharmaceutical industry to produce monoclonal antibodies, vaccines, and growth factors, etc. Cell death is an essential biological process for physiological growth and development, but it is a major problem in biopharmaceutical production in bio-industry. Cell death within bioreactor occurs due to various intracellular and extracellular stresses. These stresses negatively affect the culture longevity, overall product quality, and yield. Among all cell death types, apoptosis accounts for most of the cellular death in the bioreactor. The implementation and development of various strategies to prevent the cellular death are crucial for robust bioprocess development. Cell death during culture can be prevented or inhibited by supplementing media with specific chemicals, synthetic inhibitors, and genetic cell engineering approaches. In this review, we classified and described different types of cell death and their molecular mechanisms and summarized the cell death inhibition approaches implemented to inhibit cell death for various applications.
The escalating global population and the concomitant scarcity of protein resources have heightened the urgency to explore novel sources of protein. Insects, as a taxonomic class of organisms, are abundantly enriched with superior-quality protein and steered by this premise, they present a prodigious opportunity for human employment and scientific investigation in the realm of biologic resources. The larval stage of the domesticated silkworms, extensively cultivated for their silk, give rise to the universally consumed pupae in East Asia that augment the dietary provisions. This paper provides a comprehensive discussion of the immune-boosting, antibacterial, antitumor, and antioxidant properties of silkworm pupae proteins. Additionally, it explores the possible allergic reactions that may arise from silkworm consumption. Silkworm chrysalis protein, which contains a plethora of bioactive peptides such as AKPGVY and AAEYPA, offers a novel source of antioxidant peptides that have been shown to bolster immune function, enhance the body's capacity to fend off bacteria and viruses, reduce the risk of tumor development, and mitigate adverse effects such as free radical oxidative damage. However, it is important to note that some individuals may be susceptible to allergic reactions to silkworm pupae. Consequently, safety concerns related to the use of silkworm pupae proteins and active peptides should be taken into account.
Ferritin is an iron-binding protein composed of light-chain and heavy-chain homologs with a molecular weight of about 500 kDa. Free iron ions significantly affect reactive oxygen species (ROS) accumulation. Previous research has shown that Bombyx mori nucleopolyhedrosis virus (BmNPV) can increase ROS accumulation, activate autophagy, induce apoptosis, and upregulate the expression of B. mori ferritin heavy-chain homolog (BmFerHCH). However, the mechanism of mutual regulation between BmFerHCH and ROS-mediated autophagy and apoptosis induced by BmNPV remains unclear. In this study, we found that BmNPV induced the time-dependent accumulation of ROS in BmN cells, thereby promoting BmFerHCH expression. Interestingly, in BmFerHCH-overexpressed cells, BmNPV replication was inhibited in the first 18 h after infection but stimulated after 24 h. Further research on H2O2 or antioxidant-treated cells indicated that ROS-induced autophagy slightly increased in the early infection stage and increased BmNPV replication, while in the late stage, a large accumulation of ROS induced apoptosis and inhibited BmNPV replication. In this process, BmFerHCH inhibits BmNPV-induced ROS accumulation by chelating Fe2+. Taken together, BmFerHCH regulates ROS-mediated autophagy and apoptosis to achieve its various effects on BmNPV replication. These findings will help elucidate BmNPV-induced autophagy and apoptosis mediated by ROS and BmFerHCH, as well as the mutually fighting relationship between viruses and hosts.
The 30 K proteins are the major silkworm hemolymph proteins and are involved in a variety of physiological processes, such as nutrient and energy storage, embryogenesis, immune response, and inhibition of apoptosis. The Bm30K-15 protein is one of the 30 K proteins and is abundant in the hemolymph of fifth instar silkworm larva. We previously found that the Bm30K-15 protein can be acetylated. In the present study, we found that acetylation can improve the protein stability of Bm30K-15. Further exploration confirmed that the increase in protein stability by acetylation was caused by competition between acetylation and ubiquitination. In summary, these findings aim to provide insight into the effect of acetylation modification on the protein level and stability of the Bm30K-15 and the possible molecular mechanism of its existence in silkworm, Bombyx mori.
Research Highlights
• Bm30K-15 is acetylated and acetylation can improve its stability in silkworm.
• There is a competitive relationship between acetylation and ubiquitination.
• The competition might inhibit the ubiquitin-mediated proteasome degradation pathway.
Background
Human epidermal growth factor receptor 2 (HER2) is well-known as the therapeutic marker in breast cancer. Therefore, we evaluated anti-cancer activity of arctigenin (ATG) on in SK-BR-3 HER2-overexpressing human breast cancer cells.Methods
Cell viability and cytotoxicity were analyzed with MTT and colony-forming assay and cell cycle analysis was performed by flow cytometry. The expression and/or phosphorylation of proteins in whole cell lysate and mitochondrial fraction were analyzed by Western blotting. Cellular levels of LC3 and sequestosome 1 (SQSTM1/P62) were observed by immunofluorescence analysis.ResultsThe result showed that ATG decreased cell viability of SK-BR-3 cells in a concentration-dependent manner. Moreover, ATG increased the sub G1 population linked to the suppression of HER2/EGFR1 signaling pathway. Furthermore, ATG increased the phosphorylation of H2AX and down-regulated RAD51 and survivin expressions, indicating that ATG induced DNA damage and inhibited the DNA repair system. We also found that cleavages of caspase-7 and PARP by releasing mitochondrial cytochrome c into the cytoplasm were induced by ATG treatment for 72 h through the reduction of Bcl-2 and Bcl-xL levels in mitochondria. In contrast, the levels of LC-3 and SQSTM1/P62 were increased by ATG for 24 h through the Akt/mTOR and AMPK signaling pathway.Conclusions
Taken together, this study indicates that autophagy-linked apoptosis is responsible for the anti-cancer activity of ATG in SK-BR-3 cells, and suggests that ATG is considered a potential therapeutic for the treatment of HER2-overexpressing breast cancer.
Autophagy is an adaptive catabolic process functioning to promote cell survival in the event of inappropriate living conditions such as nutrient shortage and to cope with diverse cytotoxic insults. It is regarded as one of the key survival mechanisms of living organisms. Cells undergo autophagy to accomplish the lysosomal digestion of intracellular materials including damaged proteins, organelles, and foreign bodies, in a bulk, non-selective or a cargo-specific manner. Studies in the past decades have shed light on the association of autophagy pathways with various diseases and also highlighted the therapeutic value of autophagy modulation. Hence, it is crucial to develop effective approaches for monitoring intracellular autophagy dynamics, as a comprehensive account of methodology establishment is far from complete. In this review, we aim to provide an overview of the major current fluorescence-based techniques utilized for visualizing, sensing or measuring autophagic activities in cells or tissues, which are categorized firstly by targets detected and further by the types of fluorescence tools. We will mainly focus on the working mechanisms of these techniques, put emphasis on the insight into their roles in biomedical science and provide perspectives on the challenges and future opportunities in this field.
Acetylation is an important, highly conserved, and reversible post‐translational modification of proteins. Previously, we showed by nano‐HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K‐3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K‐3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H2O2. In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K‐3 and increase H2O2‐induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K‐3. Furthermore, the acetylation of Bm30K‐3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin‐mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori , which requires further study.
Research Highlights
• Lots of 30K proteins are acetylated in silkworm.
• Acetylation can enhance the stability and antiapoptotic activity of Bm30K‐3 by competing with its ubiquitination.
• Acetylation might regulate nutrient storage and utilization in Bombyx mori .
