A key role of L-proline metabolism in the control of stem cell identity

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. We have recently contributed to this emerging field showing that the nonessential amino acid (NEAA) L-Proline (L-Pro) acts as an epigenetic signal that promotes the conversion of mouse embryonic stem cells (ESCs) into mesenchymal-like, highly motile, invasive stem cells, which acquire metastatic potential in vivo. Despite its relevance, the molecular mechanisms underlying L-Pro -dependent control of ESC identity still remain largely unknown. Here, we provide mechanistic insights and demonstrate that ESC identity relies on a novel feedback loop that involves the L-Pro -dependent modulation of the Gcn2-Eif2?-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop places the expression of L-Pro biosynthesis genes (Aldh18a1 and Pycr1) under the control of AAR/Atf4 pathway, which is in turn under the control of L-Pro availability. This L-Pro-AAR/Atf4-Aldh18a1/Pycr1 autoregulatory loop thus maintains L-Pro as a growth limiting metabolites and generates a stress response in ESCs that restricts proliferation of tightly packed domed-like ESC colonies, and preserves ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to preserve mouse ESC identity.