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Abstract

BackgroundOne of the key challenges in biotherapeutics production is the selection of a high-producing animal cell line to maximize protein yield in cell culture. Clone selection is often a tedious process, involving rounds of selection and single cell cloning which is costly in both money and time. In an effort to increase the throughput of clone selection, we seek to identify key signatures of a highly productive cell line using an integrated genomic and proteomic platform. In our study, we analysed microarray and proteomics data generated from a characterization of two populations of CHO cells stably expressing high and low levels of green fluorescent protein (GFP). The high producer cells (HP) make 6x more GFP than the low producer cells (LP) as determined by ELISA. Comparison of transcript levels between HP and LP in the mid-exponential phase was performed using a proprietary 15k CHO cDNA microarray chip, of which 7559 genes are unique [1], while proteomic analysis on samples in the mid-exponential and stationary phases was performed using iTRAQ quantitative protein profiling technique [2]. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. ResultsFrom microarray analysis, 84 genes had a change in relative abundance of ≥ 1.5-fold, either up or down, with pvalue of ≤ 0.05. A significant number (23%) was involved in protein metabolism, transcription and RNA processing. Other major groups of genes include cell cycle regulation, signal transduction and transport. 50% of the genes had unknown functions and this could serve as a source of discovery for new and novel genes. Proteomic analysis gave 20 and 26 proteins that satisfied the cut-off criteria (≥ 1.2-fold change, 95% confidence) for the mid-exponential and stationary phase respectively. Proteins identified were mainly involved in protein metabolism, carbohydrate metabolism and transport (Figure 1). Proteome and transcriptome profiles of HP showed an up-regulation of biological processes related to protein metabolism such as protein folding (PPIB and Hyou1) and translation (Eef1a1, EIF2S3). With more protein production, genes involved in ubiquitylation (Arih1, Nedd4, Psma4, Psmc5 and Usp10) were also up-regulated to regulate misfolded proteins. Interestingly, a few of the identified genes involved in ubiquitylation have also been implicated in transcription. In particular Psmc5, a subunit of the 19S proteasome, interacts with TADs (Transcriptional Activation Domain) and general transcription factors TBP and TFIIH [3]. Key molecular chaperone genes of the UPR (unfolded protein response) pathway did not show significant differential expression, except for GRP78, an endo- plasmic reticulum molecular chaperone gene implicated in ER overload response, which was down-regulated in HP. We also found differential expression in transcription and splicing factors, which give rise to a more active transcription and mo...

Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold Potentially Targeting Fungal Succinate Dehydrogenase: Antifungal Activity and Mechanism of Action

“…Specifically, phosphoenolpyruvate carboxykinase (pckA), citrate synthase (CS), aconitate hydrogenase (ACO), isocitrate dehydrogenase (IDH2, IDH3), 2-oxoglutarate dehydrogenase (OGDH), and fumarate hydrogenase (fumC) were all downregulated, while malate dehydrogenase (MDH2) was upregulated. MDH2 is a marker enzyme derived from mitochondrial biogenesis proteins and can be expressed in the mitochondrial matrix . When MDH2 was induced by oxidative stress, the expression of MDH2 can be increased by microRNA targeting .…”

“…MDH2 is a marker enzyme derived from mitochondrial biogenesis proteins and can be expressed in the mitochondrial matrix. 31 When MDH2 was induced by oxidative stress, the expression of MDH2 can be increased by microRNA targeting. 32 The present study showed that compound SCU3038 disrupted MMP, and the dysfunction of MMP would lead to the generation and enrichment of endogenous reactive oxygen species (ROS), which in turn led to oxidative stress response, inducing microRNA to target MDH2 and upregulate its expression.…”