Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1(IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of
The GUL-1 Protein Binds Multiple RNAs Involved in Cell Wall Remodeling and Affects the MAK-1 Pathway in Neurospora crassa
The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 “core” mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway—evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1 . We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.
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- Award ID(s):
- 1916137
- PAR ID:
- 10237173
- Date Published:
- Journal Name:
- Frontiers in Fungal Biology
- Volume:
- 2
- ISSN:
- 2673-6128
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract ATF6β decreased cell growth without decreasing titer; however, knockdown ofWFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship betweenATF6β andWFS1 expression, but upregulation ofWFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown ofWFS1 had negative impacts on UPR activation and product mRNA expression, knockdown ofATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity. -
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Summary A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When
Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically‐arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically‐arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH‐1, as a key regulator of ER in the germ line. Incgh‐1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA‐binding proteins, PAB‐1, MEX‐3, and CGH‐1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line. -
null (Ed.)Abstract Background Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of Komagataella phaffii . Results By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway. Conclusion A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.more » « less
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We treated potato (Solanum tuberosum L.) plantlets with TM and performed gene expression studies to identify genome-wide changes associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). An extensive network of responses was identified, including chromatin remodeling, transcriptional reprogramming, as well as changes in the structural components of the endomembrane network system. Limited genome-wide changes in alternative RNA splicing patterns of protein-coding transcripts were also discovered. Significant changes in RNA metabolism, components of the translation machinery, as well as factors involved in protein folding and maturation occurred, which included a broader set of genes than expected based on Arabidopsis research. Antioxidant defenses and oxygen metabolic enzymes are differentially regulated, which is expected of cells that may be experiencing oxidative stress or adapting to protect proteins from oxidation. Surges in protein kinase expression indicated early signal transduction events. This study shows early genomic responses including an array of differentially expressed genes that have not been reported in Arabidopsis. These data describe novel ER stress responses in a solanaceous host.more » « less
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Abstract Like other eukaryotes, fungi use MAP kinase (MAPK) pathways to mediate cellular changes responding to external stimuli. In the past two decades, three well-conserved MAP kinase pathways have been characterized in various plant pathogenic fungi for regulating responses and adaptations to a variety of biotic and abiotic stresses encountered during plant infection or survival in nature. The invasive growth (IG) pathway is homologous to the yeast pheromone response and filamentation pathways. In plant pathogens, the IG pathway often is essential for pathogenesis by regulating infection-related morphogenesis, such as appressorium formation, penetration, and invasive growth. The cell wall integrity (CWI) pathway also is important for plant infection although the infection processes it regulates vary among fungal pathogens. Besides its universal function in cell wall integrity, it often plays a minor role in responses to oxidative and cell wall stresses. Both the IG and CWI pathways are involved in regulating known virulence factors as well as effector genes during plant infection and mediating defenses against mycoviruses, bacteria, and other fungi. In contrast, the high osmolarity growth (HOG) pathway is dispensable for virulence in some fungi although it is essential for plant infection in others. It regulates osmoregulation in hyphae and is dispensable for appressorium turgor generation. The HOG pathway also plays a major role for responding to oxidative, heat, and other environmental stresses and is overstimulated by phenylpyrrole fungicides. Moreover, these three MAPK pathways crosstalk and coordinately regulate responses to various biotic and abiotic stresses. The IG and CWI pathways, particularly the latter, also are involved in responding to abiotic stresses to various degrees in different fungal pathogens, and the HOG pathway also plays a role in interactions with other microbes or fungi. Furthermore, some infection processes or stress responses are co-regulated by MAPK pathways with cAMP or Ca 2+ /CaM signaling. Overall, functions of individual MAP kinase pathways in pathogenesis and stress responses have been well characterized in a number of fungal pathogens, showing the conserved genetic elements with diverged functions, likely by rewiring transcriptional regulatory networks. In the near future, applications of genomics and proteomics approaches will likely lead to better understanding of crosstalk among the MAPKs and with other signaling pathways as well as roles of MAPKs in defense against other microbes (biotic interactions).more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/ffunb.2021.672696
