Introduction
Mammalian cell culture, most notably Chinese Hamster Ovary (CHO) cells, are commonly used to produce recombinant proteins such as monoclonal antibodies (mAbs) by the biopharmaceutical industry for use in both therapy and diagnosis (Goulet & Atkins, 2020). Such proteins offer enormous advances in disease therapy (or diagnosis) and are products of high commercial value. Over the last several decades there have been significant advances in the efficiency of CHO cells to produce these recombinant proteins (Budge et al., 2020; Kunert & Reinhart, 2016; Sharker & Rahman, 2020; Srirangan, Loignon, & Durocher, 2020). However, the rate of protein production remains a significant bottleneck, particularly for many of the novel format based biotherapeutics now in development, and therefore there remains substantial interest in further advancing the productivity of CHO cells (Budge et al., 2020; Sharker & Rahman, 2020).
The rate of protein synthesis (mRNA Translation) is one important limiting determinant of recombinant protein production (Godfrey et al., 2017; Khoo & Al-Rubeai, 2009; O’Callaghan et al., 2010; Roobol et al., 2020; Smales et al., 2004). One key regulator of protein synthesis is the mechanistic target of rapamycin complex 1 (mTORC1) (X. Wang & Proud, 2006). mTORC1 is a protein kinase that is activated by a variety of upstream signals, most notably amino acids (Kim, Goraksha-Hicks, Li, Neufeld, & Guan, 2008; Sancak et al., 2008) and growth factors (Inoki, Li, Zhu, Wu, & Guan, 2002; B. D. Manning, Tee, Logsdon, Blenis, & Cantley, 2002), and acts as a master regulator of anabolic processes including protein synthesis (Proud, 2019) and ribosomal biogenesis (Iadevaia, Liu, & Proud, 2014; Saxton & Sabatini, 2017), both of which are critical for efficient protein production. This is achieved through phosphorylation of a number of downstream effectors such as ribosomal protein S6 kinase 1 (S6K1) (Chung, Kuo, Crabtree, & Blenis, 1992), eukaryotic elongation factor 2 kinase (eEF2K) (X. Wang et al., 2014), eIF4E binding protein 1 (4E-BP1) (Beretta, Gingras, Svitkin, Hall, & Sonenberg, 1996), and Maf1 (Michels et al., 2010), a regulator of ribosomal RNA transcription. mTORC1 signalling is thus potentially a major positive regulator of efficient, high-level production of recombinant proteins in mammalian cells. Increased phosphorylation of 4E-BP1, which permits increased translation initiation, has indeed been shown to increase production of interferon-γ (Kaur et al., 2007). We have also previously shown that the ratio of eIF4E to 4E-BP1 correlates to higher cell productivity (Jossé, Xie, Proud, & Smales, 2016). In addition to this, mTORC1 drives other anabolic pathways that contribute to cell growth and faster protein production, including lipid synthesis (Caron, Richard, & Laplante, 2015)and ribosome biogenesis (Iadevaia et al., 2014).
mTORC1 is activated by the small GTPase Rheb (Ras homologue enriched in brain) when it is in its GTP-bound form; its conversion to the inactive GDP-bound state is promoted by the tuberous sclerosis complex which includes the proteins TSC1 and TSC2, the latter acting as a GTPase-activator protein (GAP) for Rheb (Garami et al., 2003; Inoki, Zhu, & Guan, 2003; Tee, Manning, Roux, Cantley, & Blenis, 2003). In turn, the ability of TSC1/2 to impair Rheb function is inhibited by signalling events activated by hormones, mitogenic stimuli and growth factors (Huang & Manning, 2008; Inoki et al., 2002; B.D. Manning & Cantley, 2003; Zhang et al., 2003). We have recently discovered that several mutants of Rheb (which occur in certain human cancers) are resistant to the GAP activity of TSC2 and are thus ‘constitutively active’, promoting high levels of mTORC1 activity in human cells (Jianling Xie et al., 2020).
The folding, assembly and maturation of most secreted proteins occurs, with the assistance of chaperones, within the endoplasmic reticulum (ER). Homeostatic control of the ER is mediated by the unfolded protein response (UPR) (Preissler & Ron, 2019). In response to protein disequilibrium in the ER, the protein kinase RNA-like ER kinase (PERK) undergoes homodimerization and becomes active (Cui, Li, Ron, & Sha, 2011). PERK then phosphorylates eukaryotic initiation factor 2α (eIF2α) at serine-51 (Harding, Zhang, Bertolotti, Zeng, & Ron, 2000; Harding, Zhang, & Ron, 1999). eIF2α is a component of the heterotrimeric initiation factor eIF2 which is required to deliver the initiator methionyl-tRNA to the 43S preinitiation complex in order to initiate mRNA translation (Merrick & Pavitt, 2018), a process that requires the hydrolysis of eIF2-bound GTP to GDP (Kapp & Lorsch, 2004). In order to facilitate subsequent initiation events, the guanine exchange factor (GEF) eIF2B binds eIF2 and catalyses the exchange of the GDP for GTP to regenerate active eIF2.GTP. When eIF2α is phosphorylated in response to upstream stress, eIF2 is unable to be released from eIF2B and thus is no longer able to initiate translation (Bogorad, Lin, & Marintchev, 2018). This has two contrasting consequences which in combination mediate homeostasis of the ER. Firstly, it leads to global inhibition of protein synthesis, decreasing the ‘load’ of new proteins to be folded within the ER (Wek, 2018). However, secondly, certain mRNAs utilise their upstream open reading frames (uORFs) to undergo preferentially translation in response to P-eIF2α mediated inhibition of general protein synthesis (Harding, Novoa, et al., 2000). One such protein is activating transcription factor 4 (ATF4). Translation of ATF4 is thus selectively upregulated in response to phosphorylation of eIF2α. ATF4 is a transcription factor that drives the expression of genes responsible for protein homeostasis which are collectively called ER quality control genes (ERQC) (Preissler & Ron, 2019). ERQC genes consist mainly of chaperones and other protein folding genes; thus upregulation of ATF4 counters ER stress by increasing the protein folding capacity of the cell (Shaffer et al., 2004; Sriburi, Jackowski, Mori, & Brewer, 2004; M. Wang & Kaufman, 2016).
Here we show that two such Rheb mutants, T23M and E40K, drive constitutive mTORC1 signalling in CHO cells and enhance the production of recombinant protein including, importantly, its secretion from the cells. Manipulation of mTORC1 signalling by these Rheb mutants therefore has the clear potential to enhance the production of proteins of high commercial value.