## 4 Discussion  
The aim of this study was to identify new interaction partners of ERas that are involved in the maintenance of HSC quiescence. Hepatic stellate cells are not only important to sense changes within their stem cell niche, but also to contribute to inflammatory events and communicate with neighboring cells. Their role in the pathological changes during liver fibrosis is well known and connected to the continuous activation of quiescent HSCs resulting in extracellular matrix-producing myofibroblast-like cells. Therefore, identification of signaling pathways and specific proteins that are essential for HSCs quiescence or activation is necessary to understand the molecular basis of liver fibrosis and development of therapeutic approaches for patients with chronic liver diseases. In this study, we characterized the protein binding properties of the unique N-terminal extension of ERas, which is 42% identical between rat and human proteins. The MS analysis identified 20 shared proteins, pulled down with the rat and human N-terminal region (Nex), as well as 35 proteins for \(m\)Nex alone and 21 proteins for \(hs\)Nex. The molecular functions of ERas interaction partners are diverse and reach from nucleic acid binding, like the protein nucleophosmin, and structural molecule activity, like vimentin, to catalytic activities or translation regulation. Therefore, biological processes are widespread with a tendency of 29% of metabolic processes. This category includes ARG1 with its L-arginine-hydrolyzing enzymatic activity. The cellular localization of ERas binding partners are predominantly cytosolic, which coincides with the heavy and light membrane localization of ERas due to its post-translational modifications by farnesylation and palmitoylation [8; 32]. We checked the mRNA expression of four representative proteins, that differ strongly in localization and function. ARG1 was expressed strongly at day 0 cells, which are considered as quiescent HSCs. The expression of ARG1 got downregulated during activation, which gave us a first hint of the involvement of ARG1 in the maintenance of HSC quiescence. In cell biological experiments, we could confirm the coexpression, colocalization, and direct interaction of ERas and ARG1 in quiescent HSCs and also in cell-free experiments using purified proteins. Furthermore, we could confirm an active role of ARG1 when HSCs become myofibroblast-like cells, as the inhibition of arginase by nor-NOHA accelerated the activation of primary rat HSCs. Similar results were obtained after using the ODC1 inhibitor DFMO during HSC cultivation. These data suggest that not only the involvement of ARG1, but also the downstream production of polyamines are of major importance to maintain quiescence in HSCs. Polyamines can get synthesized downstream of L-ornithine by the ODC1 and further reversibly processed from putrescine to spermidine and spermine. The roles of polyamines in cells are broad and vary from cell proliferation to gene expression, ion channel regulation, and protection from oxidative damage to autophagy and regulatory aspects in immune cells [36; 37; 38; 39]. Other studies using carbon tetrachloride (CCI\({}_{4}\)) or ethanol for inducing liver injury demonstrated a protective effect of polyamines as well as a positive effect on hepatocyte proliferation [40; 41]. Interestingly, it was also reported that polyamines support the self-renewal of embryonic stem cells (ESCs) [42; 43]. ERas is highly expressed in ESCs, still ARG1 could neither be detected in the mRNA profile of embryonic stem cells, nor in mesenchymal stem cells (Supplementary Figure S10). However, polyamines can be transported from and to other cells via transporter systems and do not necessarily need to be produced and consumed in the same cell [44]. Even though the effect of polyamines on HSCs has not been described, it is tempting to speculate that the communication of HSCs and hepatocytes might include the production and exchange of polyamines to maintain liver homeostasis and regeneration after tissue injury.