Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
<i>Pichia pastoris</i> is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that <i>N</i>-acetyl-<span style="font-variant: small-caps;">l&l...
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MDPI AG
2023-03-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/7/3041 |
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| author | Yingqing Xu Zijian Geng Chengxi Yang Hongwei Zhou Yixing Wang Buayisham Kuerban Gang Luo |
| author_facet | Yingqing Xu Zijian Geng Chengxi Yang Hongwei Zhou Yixing Wang Buayisham Kuerban Gang Luo |
| author_sort | Yingqing Xu |
| collection | DOAJ |
| description | <i>Pichia pastoris</i> is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that <i>N</i>-acetyl-<span style="font-variant: small-caps;">l</span>-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC. |
| first_indexed | 2024-03-11T05:29:21Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-11T05:29:21Z |
| publishDate | 2023-03-01 |
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| record_format | Article |
| series | Molecules |
| spelling | doaj.art-051ab674c871444ca0373be2523a31fa2023-11-17T17:12:40ZengMDPI AGMolecules1420-30492023-03-01287304110.3390/molecules28073041Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion ProteinYingqing Xu0Zijian Geng1Chengxi Yang2Hongwei Zhou3Yixing Wang4Buayisham Kuerban5Gang Luo6School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, ChinaSchool of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China<i>Pichia pastoris</i> is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that <i>N</i>-acetyl-<span style="font-variant: small-caps;">l</span>-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.https://www.mdpi.com/1420-3049/28/7/3041<i>Pichia pastoris</i>autophagy<i>N</i>-acetyl-<span style="font-variant: small-caps">l-</span>cysteinecell wall |
| spellingShingle | Yingqing Xu Zijian Geng Chengxi Yang Hongwei Zhou Yixing Wang Buayisham Kuerban Gang Luo Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein Molecules <i>Pichia pastoris</i> autophagy <i>N</i>-acetyl-<span style="font-variant: small-caps">l-</span>cysteine cell wall |
| title | Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein |
| title_full | Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein |
| title_fullStr | Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein |
| title_full_unstemmed | Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein |
| title_short | Effect of <i>N</i>-acetyl-<span style="font-variant: small-caps">l</span>-cysteine on Cell Phenotype and Autophagy in <i>Pichia pastoris</i> Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein |
| title_sort | effect of i n i acetyl span style font variant small caps l span cysteine on cell phenotype and autophagy in i pichia pastoris i expressing human serum albumin and porcine follicle stimulating hormone fusion protein |
| topic | <i>Pichia pastoris</i> autophagy <i>N</i>-acetyl-<span style="font-variant: small-caps">l-</span>cysteine cell wall |
| url | https://www.mdpi.com/1420-3049/28/7/3041 |
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