Transient vitamin B5 starving improves mammalian cell homeostasis and protein production

Pourcel, Lucille; Buron, Flavien; Garcia, Fanny; Delaloix, Margaux-Sarah; Fourn, Valérie Le; Girod, Pierre‐Alain; Mermod, Nicolas

doi:10.1016/j.ymben.2020.03.008

Cited by 18 publications

(18 citation statements)

References 29 publications

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“…Ulcerative colitis is usually accompanied by the accumulation of colon stem cells and undifferentiated transit expanded cells at the base of the crypt (Papapietro et al, 2013), resulting in the inhibition of peroxisome proliferator-activated receptor (PPAR) synthesis in epithelial cells (Dubuquoy et al, 2003;Litvak et al, 2018). Pantothenic acid has been shown to continuously activate PPARs (Pourcel et al, 2020), thus reducing the risk of increased colon permeability (Ponferrada et al, 2007). However, pantothenic acid is mainly involved in metabolism in the form of coenzyme A. Coenzyme A is a ubiquitous cofactor that plays an irreplaceable role in carboxylic acid metabolism (Leonardi et al, 2005), and short-chain fatty acids have long been known to inhibit histone deacetylase and activate G-proteincoupled receptors to regulate the intestinal health (Sun et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Lactobacillus pentosus Increases the Abundance of Akkermansia and Affects the Serum Metabolome to Alleviate DSS-Induced Colitis in a Murine Model

Yan

et al. 2020

Front. Cell Dev. Biol.

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Lactobacillus pentosus has the beneficial function of regulating the host's immune system and plays an indispensable role in intestinal health. The purpose of this study was to investigate the specific mechanism by which L. pentosus relieves dextran sulfate sodium (DSS) induced ulcerative colon inflammation. We randomly divided 24 mice into three groups, which were administered either a basic diet, drinking water with 2.5% DSS (DSS), or drinking water with 2.5% DSS and intragastric administration of L. pentosus (DSS + L. pentosus). DSS was added to the drinking water on days 8 to 12, and L. pentosus was administered on days 12 to 19. Serum was collected for metabolomic analysis, colon length and weight were measured, and colon contents were collected to detect microbial structural composition. Compared with the DSS group, the DSS + L. pentosus group had significantly higher levels of indolepyruvate and pantothenic acid in the serum and significantly lower levels of 3,4-dimethyl-5pentyl-2-furannonanoic acid and 5-oxo-6-trans-leukotriene B4. Moreover, compared with the other two groups, the DSS + L. pentosus group had a significantly greater abundance of Akkermansia. The abundance of Akkermansia was positively correlated with indolepyruvate and pantothenic acid levels. Therefore, L. pentosus can interact with Akkermansia to increase its abundance in the intestinal tract. This results in the production of metabolites that are beneficial for the regulation of intestinal immunity, thereby alleviating DSS-induced ulcerative colon inflammation.

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Section: Discussionmentioning

confidence: 99%

Lactobacillus pentosus Increases the Abundance of Akkermansia and Affects the Serum Metabolome to Alleviate DSS-Induced Colitis in a Murine Model

Yan

et al. 2020

Front. Cell Dev. Biol.

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“…Gene induction after B5 selection may be caused either by B5 starvation occurring during the selective process, as found in a previous study (Pourcel et al, 2020), by the overexpression of SLC5A6 itself, as it mediates higher vitamin B5 intake into the cell (Figure 1d), or by a combination of both effects. B5 is an .…”

Section: Effects Of Cytoskeleton-related Genes On Therapeutic Protementioning

confidence: 57%

“…Fed-batch performance evaluation, IgG cell surface staining, IgG cell secretion assay, and vitamin B5 metabolite quantification, were performed as previously described (Pourcel et al, 2020).…”

Section: Analyses Of Stable Cell Pools and Cell Linesmentioning

confidence: 99%

“…We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.actin polymerization, CHO cells, cytoskeleton regulation, recombinant protein production 1 | INTRODUCTION Improving recombinant therapeutic protein manufacturing using mammalian Chinese hamster ovary (CHO) cell hosts is a major challenge for biopharmaceutical companies. Thus, many research are currently ongoing to unravel and overcome the remaining unknown metabolic bottlenecks of these cells for high-level protein production.We previously described how the deprivation of vitamin B5 in CHO cell culture medium, coupled to the cotransfection of an expression vector for a vitamin B5 transport protein with the gene of interest, allowed the identification of cell variants that are capable of expressing at very high levels recombinant therapeutic proteins (Pourcel et al, 2020). However, the cellular properties that allow very high-level recombinant protein secretion remain unknown.In this study, we wished to identify specific gene expression alterations that accompany efficient therapeutic protein expression, with the hope to construct CHO cell line derivatives that would be permanently more efficient for production.…”

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confidence: 99%

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Influence of cytoskeleton organization on recombinant protein expression by CHO cells

Pourcel

Buron

Arib³

et al. 2020

Biotech & Bioengineering

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In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.

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“…Clones producing recombinant proteins at the highest levels were evaluated for growth and production performances in 10 days fed-batch 20-ml cultures to select the most favorable clones according to cell productivity, cell-line stability, and product quality attributes. Clones producing the IFN-b1a following vitamin B5 selection were described previously (Pourcel, Buron, Garcia, et al, 2020).…”

Section: Cho Cell Line Developmentmentioning

confidence: 99%

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

Berger

Fourn²,

Masternak

et al. 2020

Biotech & Bioengineering

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Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy‐to‐express antibody, whereas Erp27 and Erp57 co‐overexpression increased cell density, viability, and the yield of difficult‐to‐express proteins. Foxa1 overexpression increased cell density, cell viability, and easy‐ and difficult‐to‐express protein yields, whereas it decreased reactive oxygen species late in fed‐batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult‐ and/or easy‐to‐express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

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Transient vitamin B5 starving improves mammalian cell homeostasis and protein production

Cited by 18 publications

References 29 publications

Lactobacillus pentosus Increases the Abundance of Akkermansia and Affects the Serum Metabolome to Alleviate DSS-Induced Colitis in a Murine Model

Lactobacillus pentosus Increases the Abundance of Akkermansia and Affects the Serum Metabolome to Alleviate DSS-Induced Colitis in a Murine Model

Influence of cytoskeleton organization on recombinant protein expression by CHO cells

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

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