| 1. |
- Chen, Xin, 1980, et al.
(author)
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Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast
- 2022
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In: Data in Brief. - : Elsevier BV. - 2352-3409. ; 42
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Journal article (peer-reviewed)abstract
- The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-beta peptides (A beta 42) were accumulated [1,2] . We have previously identified suppressors of A beta 42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4] . The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-beta Toxicity Improve Recombinant Protein Produc-tion in yeast by Reducing Oxidative Stress and Tuning Cellu-lar Metabolism", by Chen et al. [5] . (C) 2022 The Author(s). Published by Elsevier Inc.
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| 2. |
- Chen, Xin, 1980, et al.
(author)
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Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism
- 2022
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324
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Journal article (peer-reviewed)abstract
- High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
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| 3. |
- Chen, Xin, 1980, et al.
(author)
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FMN reduces Amyloid-beta toxicity in yeast by regulating redox status and cellular metabolism
- 2020
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Journal article (peer-reviewed)abstract
- Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-beta peptides (A beta) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of A beta 42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce A beta 42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH+NAD(+)) and NADPH/(NADPH+NADP(+)) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and alpha-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of A beta 42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.Saccharomyces cerevisiae is a model organism to study proteins involved in neurodegeneration. Here, the authors performed a yeast genome-wide synthetic genetic interaction array (SGA) to screen for toxicity modifiers of A beta 42 and identify riboflavin kinase and its metabolic product flavin mononucleotide as modulators that alleviate cellular A beta 42 toxicity, which is supported by further experimental analyses.
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| 4. |
- Chen, Xin, 1980, et al.
(author)
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Graphene Oxide Attenuates Toxicity of Amyloid-β Aggregates in Yeast by Promoting Disassembly and Boosting Cellular Stress Response
- 2023
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In: Advanced Functional Materials. - 1616-3028 .- 1616-301X. ; 33:45
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Journal article (peer-reviewed)abstract
- Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, with the aggregation of misfolded amyloid-β (Aβ) peptides in the brain being one of its histopathological hallmarks. Recently, graphene oxide (GO) nanoflakes have attracted significant attention in biomedical areas due to their capacity of suppressing Aβ aggregation in vitro. The mechanism of this beneficial effect has not been fully understood in vivo. Herein, the impact of GO on intracellular Aβ42 aggregates and cytotoxicity is investigated using yeast Saccharomyces cerevisiae as the model organism. This study finds that GO nanoflakes can effectively penetrate yeast cells and reduce Aβ42 toxicity. Combination of proteomics data and follow-up experiments show that GO treatment alters cellular metabolism to increases cellular resistance to misfolded protein stress and oxidative stress, and reduces amounts of intracellular Aβ42 oligomers. Additionally, GO treatment also reduces HTT103QP toxicity in the Huntington's disease (HD) yeast model. The findings offer insights for rationally designing GO nanoflakes-based therapies for attenuating cytotoxicity of Aβ42, and potentially of other misfolded proteins involved in neurodegenerative pathology.
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| 5. |
- Chen, Xin, 1980, et al.
(author)
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UBB+1 reduces amyloid-beta cytotoxicity by activation of autophagy in yeast
- 2021
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In: Aging. - : Impact Journals, LLC. - 1945-4589. ; 13:21, s. 23953-23980
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Journal article (peer-reviewed)abstract
- UBB+1 is a mutated version of ubiquitin B peptide caused by a transcriptional frameshift due to the RNA polymerase II "slippage". The accumulation of UBB+1 has been linked to ubiquitin-proteasome system (UPS) dysfunction and neurodegeneration. Alzheimer's disease (AD) is defined as a progressive neurodegeneration and aggregation of amyloid-beta peptides (A beta) is a prominent neuropathological feature of AD. In our previous study, we found that yeast cells expressing UBB+1 at lower level display an increased resistance to cellular stresses under conditions of chronological aging. In order to examine the molecular mechanisms behind, here we performed genome-wide transcriptional analyses and molecular/cellular biology assays. We found that low UBB+1 expression activated the autophagy pathway, increased vacuolar activity, and promoted transport of autophagic marker ATG8p into vacuole. Furthermore, we introduced low UBB+1 expression to our humanized yeast AD models, that constitutively express A beta 42 and A beta 40 peptide, respectively. The co-expression of UBB+1 with A beta 42 or A beta 40 peptide led to reduced intracellular A beta levels, ameliorated viability, and increased chronological life span. In an autophagy deficient background strain (atg1 Delta), intracellular A beta levels were not affected by UBB+1 expression. Our findings offer insights for reducing intracellular A beta toxicity via autophagydependent cellular pathways under low level of UBB+1 expression.
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| 6. |
- Meza, Eugenio, 1975, et al.
(author)
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Development of a method for heat shock stress assessment in yeast based on transcription of specific genes
- 2021
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In: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 38:10, s. 549-565
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Journal article (peer-reviewed)abstract
- All living cells, including yeast cells, are challenged by different types of stresses in their environments and must cope with challenges such as heat, chemical stress, or oxidative damage. By reversibly adjusting the physiology while maintaining structural and genetic integrity, cells can achieve a competitive advantage and adapt environmental fluctuations. The yeast Saccharomyces cerevisiae has been extensively used as a model for study of stress responses due to the strong conservation of many essential cellular processes between yeast and human cells. We focused here on developing a tool to detect and quantify early responses using specific transcriptional responses. We analyzed the published transcriptional data on S. cerevisiae DBY strain responses to 10 different stresses in different time points. The principal component analysis (PCA) and the Pearson analysis were used to assess the stress response genes that are highly expressed in each individual stress condition. Except for these stress response genes, we also identified the reference genes in each stress condition, which would not be induced under stress condition and show stable transcriptional expression over time. We then tested our candidates experimentally in the CEN.PK strain. After data analysis, we identified two stress response genes (UBI4 and RRP) and two reference genes (MEX67 and SSY1) under heat shock (HS) condition. These genes were further verified by real-time PCR at mild (42°C), severe (46°C), to lethal temperature (50°C), respectively.
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| 7. |
- Wang, Yanyan, 1989, et al.
(author)
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CRISPR/Cas9-mediated point mutations improve alpha-amylase secretion in Saccharomyces cerevisiae
- 2022
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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Journal article (peer-reviewed)abstract
- The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved alpha-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve alpha-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion. Systematic characterization of point mutations from evolved strains using CRISPR/Cas9 technology revealed a set of gene alterations that improved recombinant protein secretion in Saccharomyces cerevisiae.
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| 8. |
- Wang, Yanyan, 1989, et al.
(author)
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Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion
- 2021
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 20:1
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Journal article (peer-reviewed)abstract
- Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of posttranslational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.
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