Cargando…

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein

Pichia pastoris 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 N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion pro...

Descripción completa

Detalles Bibliográficos
Autores principales: Xu, Yingqing, Geng, Zijian, Yang, Chengxi, Zhou, Hongwei, Wang, Yixing, Kuerban, Buayisham, Luo, Gang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095845/
https://www.ncbi.nlm.nih.gov/pubmed/37049804
http://dx.doi.org/10.3390/molecules28073041
_version_ 1785024180390985728
author Xu, Yingqing
Geng, Zijian
Yang, Chengxi
Zhou, Hongwei
Wang, Yixing
Kuerban, Buayisham
Luo, Gang
author_facet Xu, Yingqing
Geng, Zijian
Yang, Chengxi
Zhou, Hongwei
Wang, Yixing
Kuerban, Buayisham
Luo, Gang
author_sort Xu, Yingqing
collection PubMed
description Pichia pastoris 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 N-acetyl-l-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.
format Online
Article
Text
id pubmed-10095845
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-100958452023-04-13 Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein Xu, Yingqing Geng, Zijian Yang, Chengxi Zhou, Hongwei Wang, Yixing Kuerban, Buayisham Luo, Gang Molecules Article Pichia pastoris 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 N-acetyl-l-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. MDPI 2023-03-29 /pmc/articles/PMC10095845/ /pubmed/37049804 http://dx.doi.org/10.3390/molecules28073041 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xu, Yingqing
Geng, Zijian
Yang, Chengxi
Zhou, Hongwei
Wang, Yixing
Kuerban, Buayisham
Luo, Gang
Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title_full Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title_fullStr Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title_full_unstemmed Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title_short Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein
title_sort effect of n-acetyl-l-cysteine on cell phenotype and autophagy in pichia pastoris expressing human serum albumin and porcine follicle-stimulating hormone fusion protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095845/
https://www.ncbi.nlm.nih.gov/pubmed/37049804
http://dx.doi.org/10.3390/molecules28073041
work_keys_str_mv AT xuyingqing effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT gengzijian effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT yangchengxi effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT zhouhongwei effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT wangyixing effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT kuerbanbuayisham effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein
AT luogang effectofnacetyllcysteineoncellphenotypeandautophagyinpichiapastorisexpressinghumanserumalbuminandporcinefolliclestimulatinghormonefusionprotein