title: Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress.
abstract: BACKGROUND
  The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).
  
  
  RESULTS
  HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.
  
  
  CONCLUSIONS
  Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Göteborg, Sweden.
authors.forename: Keith E J
authors.initials: KE
authors.lastname: Tyo
authors.forename: Zihe
authors.initials: Z
authors.lastname: Liu
authors.forename: Dina
authors.initials: D
authors.lastname: Petranovic
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D001692:Biological Transport; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress; D011384:Proinsulin / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D000516:alpha-Amylases / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004220:Disulfides; C091341:HAC1 protein, S cerevisiae; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D011384:Proinsulin; D000516:alpha-Amylases; D005978:Glutathione