###### Abstract  
Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence. orgnagel 1, ARG1; embryonic stem cell-expressed Ras; ERas; hepatic stellate cells; quiescence; iNOS; L-arginine; L-ornithine; polyamines; spermidine; spermine; urea cycle +
Footnote †: journal: Accepted: 27 January 2022  
that reside in close contact with sinusoidal endothelial cells in the space of Disse [9]. This unique space in liver sinusoids is bordered by endothelial cells and hepatocytes providing a niche that helps to sustain HSC quiescence [10]. Once activated, HSCs show typical characteristics and functions of mesenchymal stem cells and have therefore been classified as such [11]. A mechanism that has since been described for pericytes of other organ systems as well [12]. An intact stem cell niche is crucial for the maintenance of stemness, differentiation and developmental fate decisions of stem cells [10; 13]. In a normal, healthy liver, HSCs represent 5-8% of the total liver cells and store about 85% of the body's vitamin A as retinyl palmitate in membrane-coated vesicles [9]. The expression of neural and mesodermal markers, i.e., glial fibrillary protein (GFAP) and desmin, are known to be displayed in a quiescent phenotype. Following liver injury, quiescent HSCs (qHSCs) activate and develop into proliferative and contractile myofibrroblast-like cells (aHSCs), revealing profibrogenic transcriptional properties and accounting for extracellular matrix accumulation [14; 15]. Activated HSCs show downregulation of GFAP and ERas, upregulation of \(\alpha\)-smooth muscle actin (\(\alpha\)-SMA) and collagen type I, as well as loss of lipid droplets [8; 10]. Arginase (EC 3.5.3.1.) is a manganese-containing enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It is a key enzyme of the hepatic urea cycle but is also expressed in extrahepatic tissues lacking a complete urea cycle. There are two paralogs that differ in expression, regulation, and localization [16]. The nonmitochondrial, cytosolic enzyme, arginase 1 (ARG1), is the predominant paralog in the liver and red blood cells [17], whereas the mitochondrial arginase 2 is mainly expressed in extrahepatic tissues [16; 18; 19; 20]. L-arginine is not only a substrate for arginase, but can alternatively be converted to nitric oxide (NO) and L-citrulline by nitric oxide synthases (NOS). Thus, one biological function of arginase in extra-hepatic organs lies in the regulation of NO synthesis, by competing with NOS for the common substrate L-arginine [21] and also participates in numerous inflammatory diseases by the downregulation of NOS activity, the induction of fibrosis and tissue regeneration [22].