Is the absence of alpha-helix 2 in the appendant structure region the major contributor to structural instability of human cystatin C?

Zhou, Xuejie; Lu, Xiaoping; Qin, Shuzhen; Xu, Linan; Chong, Xiaoying; Liu, Junqing; Yan, Pingyu; Sun, Rui; Hurley, Ian P.; Jones, Gary W.; Wang, Qiuyu; He, Jing

doi:10.1080/07391102.2018.1552625

Cited by 2 publications

(4 citation statements)

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“…Extracellular protein samples were obtained as previously described ( Zhou et al, 2019 ). Intracellular protein samples were extracted from yeast cells after treatment with Yeast Protein Extraction Reagent (Takara, Dalian, China).…”

Section: Methodsmentioning

confidence: 99%

“…Intracellular protein samples were extracted from yeast cells after treatment with Yeast Protein Extraction Reagent (Takara, Dalian, China). SDS-PAGE and western blotting were carried out following the procedure described previously ( Zhou et al, 2019 ). The protein bands in one gel were visualized by staining the gel with PAGE Gel Silver Staining Kit (Takara, Beijing, China) whereas the protein bands in the other gel were transferred to a PVDF membrane (Millipore, MA, USA) for western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, the α-helix 2-truncated mutant (ΔW) with a deletion at residues 77–85 was constructed as the unstable cC model protein. Based on our previous results, the secreted amount of ΔW is much lower than that of WT cC or I66Q when expressed in P. pastoris , indicating that the absence of α-helix 2 in the AS region may be one of the factors contributing to the structural instability of HCC ( Zhou et al, 2019 ).…”

Section: Introductionmentioning

confidence: 92%

“…Co-expression of PDI has been used to improve the expression of heterologous proteins in P. pastoris by overcoming the burden of protein folding and secretion (Inan et al, 2006;Navone et al, 2021a). In our previous study, the overexpression of PDI in P. pastoris GS115 strains was found to significantly increase the expression of cC (Zhou et al, 2019). On this basis, three representative proteins with different stabilities (WT cC and its two mutants I66Q and W) (Figure 1) were used as model proteins to screen for factors other than the ER quality control system represented by the molecular chaperone PDI that could influence the expression of foreign proteins that may not be properly folded in P. pastoris.…”

Section: Introductionmentioning

confidence: 98%

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Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris

Niu

Cui

et al. 2022

Front. Microbiol.

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IntroductionThe industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability.MethodsWild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed.Results and discussionThe coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability.ConclusionThese findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%