The Multifaceted Roles of Proline in Cell Behavior

Herein, we review the multifaceted roles of proline in cell biology. This peculiar cyclic imino acid is: (i) A main precursor of extracellular collagens (the most abundant human proteins), antimicrobial peptides (involved in innate immunity), salivary proteins (astringency, teeth health) and cornifins (skin permeability); (ii) an energy source for pathogenic bacteria, protozoan parasites, and metastatic cancer cells, which engage in extracellular-protein degradation to invade their host; (iii) an antistress molecule (an osmolyte and chemical chaperone) helpful against various potential harms (UV radiation, drought/salinity, heavy metals, reactive oxygen species); (iv) a neural metabotoxin associated with schizophrenia; (v) a modulator of cell signaling pathways such as the amino acid stress response and extracellular signal-related kinase pathway; (vi) an epigenetic modifier able to promote DNA and histone hypermethylation; (vii) an inducer of proliferation of stem and tumor cells; and (viii) a modulator of cell morphology and migration/invasiveness. We highlight how proline metabolism impacts beneficial tissue regeneration, but also contributes to the progression of devastating pathologies such as fibrosis and metastatic cancer.


INTRODUCTION
In 1900, Richard M. Willstätter reported the synthesis of (S)pyrrolidine-2-carboxylic acid, better known as L-Pro. Town (1928) reported the purification of L-Pro from gliadin proteins, and Levine (1959) reported that nitrous acid destroys all amino acids apart from L-Pro in hydrolyzed gelatins, and highlighted its unusual structure. L-Pro is a small (115.13 g/mol), cyclic, non-polar, non-toxic, odorless, sweet-tasting imino acid, with unique physicochemical proprieties and numerous biotechnological applications (Figure 1). For instance, acting as an enantioselective organocatalyst, L-Pro makes possible the synthesis of therapeutically active enantiopure drugs (Table 1). Moreover, acting as a chemical chaperone, L-Pro can prevent protein aggregation/fibrillation, and is therefore used to stabilize monoclonal antibodies, to generate protein crystals (Table 1), and for the cryopreservation of biological specimens, including stem cells and oocytes ( Table 1). Due to its peculiar cyclic structure, its metabolism relies on specific enzymes. For instance, in mammalian cells L-Pro is synthesized from Lglutamate in a two-step intramitochondrial process catalyzed by aldehyde dehydrogenase 18 family member A1 (ALDH18A1) and pyrroline-5-carboxylate reductase 1 (PYCR1) enzymes (Figure 1), whereas it is oxidized to L-glutamate in a two-step intramitochondrial process catalyzed by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) enzymes (Figure 1).

PROLINE IN EXTRACELLULAR MATRIX PRODUCTION
L-Proline residues constitute nearly 6% of the human proteome, mainly concentrated in L-Pro-rich proteins, with up to 1 × 10 4 L-Pro-rich motifs/stretches occurring in 1.8 × 10 4 human proteins (Morgan and Rubenstein, 2013;Mandal et al., 2014). In addition to a high L-Pro content (up to 50% of total residues), L-Pro-rich peptides/proteins share extracellular localization (secreted proteins), a dedicated translation factor (EIF5A), and a requirement for timely L-Pro-tRNA loading (Doerfel et al., 2013;Gutierrez et al., 2013;Wu et al., 2020;Faundes et al., 2021). Free L-Pro is derived from dietary sources (animal collagens or vegetable extensins) or from de novo biosynthesis (Figure 1), which relies on mitochondrial generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Tran et al., 2021;Zhu et al., 2021). Why so many extracellular proteins are rich in L-Pro is a fascinating question; L-Pro residues destabilize α-helices and β-sheets protein secondary structures, enables turns and poly-Pro helices, and are major 'disorder-promoting' residues in intrinsically disordered proteins (Theillet et al., 2013;Alderson et al., 2018;Mateos et al., 2020).

Matrix Collagens
Collagens constitute ∼30% of total human proteins (Smith and Rennie, 2007), and are secreted by cells of CTs such as bone, cartilage, tendon, ligament, and interconnected fluid-filled CTs (Benias et al., 2018) that support and connects all other tissues (epithelial, muscular, etc.). Collagen synthesis is highly dependent on L-Pro availability (∼170 µM in plasma) (Psychogios et al., 2011), and inherited mutations in ALDH18A1 or PYCR1 (de novo L-Pro biosynthesis) are a cause of abnormal CT development ( Table 2). Extrinsic (dietary) L-Pro is essential during adult life to preserve bone density in a mice model of osteoporosis (Nam et al., 2016), collagen deposition in rats, pigs, chickens and fish (Li and Wu, 2018;He et al., 2021), and L-Pro homeostasis in humans (Jaksic et al., 1990;Bertolo and Burrin, 2008).

Cornified Cell Envelope
Skin is the largest organ of the human body, and it protects internal tissues/organs from water and heat loss, physicochemical insults (e.g., UV light), and microbial attack. Cornifins (or SPRRs) are cross-bridging L-Pro-rich proteins of the cell envelope (Marvin et al., 1992;Steinert et al., 1998a,b), a 5-15 nm thick layer of proteins deposited in epidermis corneocytes (Figure 2). Cornifins are markers of psoriasis syndrome (Luo et al., 2020) and are induced in some tumors (Deng et al., 2020;Sasahira et al., 2021).

PROLINE IN ENERGY PROVISION
Cells obtain energy/ATP through oxidation of glucose, fatty acids or L-glutamine. However, some cells obtain energy via oxidation of L-Pro in a three-step process (see Figure 3) that converts L-Pro into α-KG, a Krebs cycle intermediate (Tanner et al., 2018). Up to 30 ATP equivalents per L-Pro molecule can sustain the growth of dissimilar cell types, from bacteria to insect muscle cells and human cancer cells (Servet et al., 2012;Nishida et al., 2016). Of note, human genetic defects in L-Pro oxidation are not associated with any developmental deficiency, suggesting that any normal cell type in the human body is strictly reliant on L-Pro energy.

Cancer Cells
Pancreatic and mammary tumor tissues are full of collagens, providing a large reservoir of free L-Pro (Linder et al., 2001;Barcus et al., 2017). Prolyl-specific peptidases are induced in cancer cells and can release L-Pro-rich peptides and free L-Pro in their microenvironment by degrading ECM collagens (Figure 3; Pure and Blomberg, 2018). For instance, free L-Pro is accumulated in esophageal carcinoma tissue, where it reaches significantly higher levels than in neighboring normal tissues (Sun et al., 2019). Free L-Pro is transported inside cancer cells, where it can be used for anabolic and catabolic purposes. Indeed, PDAC cancer cells (Olivares et al., 2017), colorectal cancer cells (Liu et al., 2012a), and transformed mammary epithelial cells (MCF10A H-Ras V 12 ) growing as 3D spheroids (Elia et al., 2017) use L-Pro to obtain energy/ATP (Figure 3). L-Pro is also used to produce new collagens (L-Pro recycling), and, eventually, to alter the ECM composition/stiffness (D'Aniello et al., 2020).

Insect Flight Muscle
Flight is one of the highest ATP/energy-requiring processes in animals, and the muscle cells involved can make use of different energy sources including carbohydrates (e.g., honeybee Apis mellifera) and fatty acids (e.g., butterflies) (Bursell, 1975;Candy et al., 1997). Some insects, such as Locusta migratoria, Bombus impatiens (bumblebee), Vespula vulgaris and Glossina diptera, oxidize L-Pro to power flight (Figure 3; Teulier et al., 2016). L-Pro supports flight muscle cells of Aedes aegypti mosquitoes that feed on blood and can obtain free L-Pro from the hydrolysis of blood proteins and/or from alanine in the fat body (Goldstrohm et al., 2003;Scaraffia and Wells, 2003;Mazzalupo et al., 2016). Indeed, free L-Pro is abundant in the hemolymph of adult female mosquitoes and other insects such as Diaphorina citri, the vector of Candidatus Liberibacter asiaticus (huanglongbing) (Killiny et al., 2017).

Polyamine and Citrate Precursors
Some cells use the carbon skeleton of L-Pro to synthesize Lornithine and L-arginine. For instance, in the gut of neonates, L-glutamate to pyrroline-5-carboxylate conversion is negligible,  Vesicles of sarcoplasmic reticulum from lobster muscle L-Proline (more effective than glycerol or DMSO) Rudolph and Crowe (1985) Applications in: 1 pharmaceutical industry, 2 pharmacological therapy, and 3 biomedical research, regenerative medicine.
Frontiers in Cell and Developmental Biology | www.frontiersin.org hence dietary L-Pro is the only source of L-arginine (Tomlinson et al., 2011a,b). In motile human spermatozoa, L-Pro is the precursor of polyamines such as putrescine, spermidine and spermine (Figure 3; Wu et al., 2005Wu et al., , 2008, which are deregulated in hyper-proliferative cancer cells (Bachmann and Geerts, 2018), and thus a potential target for therapeutic anticancer intervention (Murray-Stewart et al., 2016). The three-step L-Pro to α-KG conversion is also activated to generate Krebs-derived metabolic intermediates. For instance, cells of mouse retinal pigment epithelium use L-Pro to synthesize and export citrate, which is consumed by the outer retina (Figure 3; Chao et al., 2017;Yam et al., 2019;Du et al., 2021).

PROLINE IN ANTISTRESS RESPONSE
Living cells are subjected to a fluctuating environment involving transient or continuous changes in physicochemical parameters such as temperature, humidity and UV radiation. For instance, humans renal and corneal cells are exposed to discontinuous but substantial variations in osmolality/salinity. To prevent the detrimental effects of such harmful environmental imbalances, cells utilize adaptive mechanisms, including accumulation of highly soluble non-toxic osmolytes and chemical chaperones (protein stabilizers) such as L-Pro. Of course, living cells can tolerate extensive accumulation of L-Pro (up to a 100-fold increase) without suffering of the ionic imbalances induced by accumulation of inorganic osmolytes (e.g., Na + , K + , Mg +2 or Ca +2 salts).

Radical Scavenging
L-Proline protects various human cells such as HEK293, HeLa, HepG2, Jurkat, BJAB, WM35, skin keratinocytes and fibroblasts against ROS-mediated oxidative stress (Figure 4; Wondrak et al., 2005;Krishnan et al., 2008;Natarajan et al., 2012). Of note, the five-membered ring of L-Pro molecule, known as pyrrolidine or tetrahydropyrrole, quenches hydroxyl radicals ( · OH) (Signorelli, 2016). In plants L-Pro accumulates in response to oxidative compounds (Yang et al., 2009;Ben Rejeb et al., 2015), and  Cg-Prp (37 aa  Antheraea mylita Antibacterial, antifungal Cell membrane damage, cell lysis Chowdhury et al. (2021) Frontiers in Cell and Developmental Biology | www.frontiersin.org FIGURE 2 | Proline in extracellular matrix production. Proline is a crucial building block of antimicrobial peptides, salivary proteins, epidermal cornifins, interstitial collagens, and plant nodulins. These proteins are all rich in proline residues (with up to 50% of total amino acids) and are all secreted in the extracellular space. In addition to shape cell/tissue microenvironment/architecture (fibrillar collagens), proline-rich proteins contribute to innate immunity (antibiotic activity) by inhibiting bacterial protein synthesis (top left), to diet selection (astringency) by binding polyphenolic tannis (top right) and, to teeth health by inducing enamel mineralization and preventing bacterial attacks (top right), to selective permeation (barrier of water, O 2 ) by nodulins in N 2 fixing root nodules of leguminous plants (middle left), and by cornifins in skin (middle right), and to signaling mechanical forces (ECM stiffness). The accumulation of interstitial collagens leads to pathological fibrosis and occurs in different tumoral tissues (bottom right).

ER Stress Relief
At a molecular level, various stressful conditions (e.g., suboptimal temperature, high salinity and oxidative agents) can destabilize the structure and conformation of cellular proteins and other macromolecules. Thus, the accumulation of L-Pro (chemical In the tumor microenvironment, collagens degradation enzymes such as fibroblast activation protein (FAP), and prolyl endopeptidase PREP) release proline-rich peptides and free proline, which after internalization can serves to produce ATP and/or new collagens. Intramitochondrial enzymes involved in L-proline (L-Pro) oxidation, namely proline dehydrogenase (PRODH) and the pyrroline-5-carboxylate dehydrogenase (P5CDH), and glutamate dehydrogenase (GDH), are indicated. chaperone) represents a convergent response of cells aimed at inhibiting the formation of unfolded/misfolded protein aggregates. In this context, induction of ATF4 expression (Figure 4), and subsequent enhancement of the transcription of genes involved in L-Pro uptake (SLC38A2) and biosynthesis (ALDH18A2, PYCR1) can contribute to intracellular L-Pro accumulation (D'Aniello et al., 2015). By stabilizing protein folding and/or promoting protein refolding, L-Pro can avoid and/or relieve ER stress. Schafer et al. (1962) reported a link between hyperprolinemia (HP), characterized by high levels of plasmatic L-Pro, and neuronal dysfunction in human patients. It later emerged that different forms of hereditary human HP (type I or II) are associated with defects in L-Pro oxidation/degradation (Geraghty et al., 1998;Jacquet et al., 2002). Indeed, ectopic expression of PRODH in glioblastoma cells reduces the level of L-Pro FIGURE 4 | Proline in anti-stress response. Proline accumulation is an evolutionary conserved cell defense mechanism against stressful environments; by quenching hydroxyl radicals ( · OH), protects the cells from ROS oxidations (top left); as osmolyte avoids high salinity-mediated cell shrinkage (top right), as well as the formation of ice crystal, and thus protects many organisms (yeast, plants, overwinter insect) from cell disruption by freezing (middle right). As a chemical chaperone avoids protein denaturation and thus the accumulation of misfolded proteins (middle left), which are potent inducers of a molecular response that involves the protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylation of eukaryotic initiation factor 2 (EIF2), and eventually, the translation of activating transcription factor 4 (ATF4) (bottom left); ATF4 in turn, induces the expression of solute carrier family 38 member 2 (SLC38A2), growth arrest and DNA damage-inducible protein (GADD34), aldehyde dehydrogenase 18 family member a1 (ALDH18A1) and pyrroline-5-carboxylate reductase 1 (PYCR1) (bottom right). Intracellular proline accumulation through proline uptake and de novo proline biosynthesis (center) can contribute to stress alleviation. (Cappelletti et al., 2018). Free L-Pro can interfere with excitatory presynaptic transmission, and therefore normal neuronal activity in the central nervous system (CNS) (Shafqat et al., 1995;Velaz-Faircloth et al., 1995;Wyse and Netto, 2011). Of note, the psychostimulant methamphetamine induces L-Pro synthesis in human neuroblastoma cells .
Human patients with genetic defects in PRODH (HPI, L-Pro levels up to 10-fold higher than normal) or in P5CDH (ALDH4A1; HPII, L-Pro levels up to 15-fold higher and P5C excretion) suffer schizoaffective disorders and schizophrenia ( Table 2; Liu et al., 2002;Bender et al., 2005;Raux et al., 2007;Clelland et al., 2011;Nagaoka et al., 2020). At high levels, L-Pro can be oxidized/converted into the neurotransmitter Lglutamate, which is associated with schizophrenia ( Figure 5). Excess L-glutamate disturbs synaptic transmission and can destroy neurons, a process known as excitotoxicity (Nadler et al., 1988;Cohen and Nadler, 1997). Moreover, acting as a GABA mimetic inhibitor of the GAD enzyme, L-Pro can reduce the synthesis the GABA neurotransmitter, thereby provoking synaptic dysfunction (Figure 5; Crabtree et al., 2016). Of note, L-Pro antagonizes GABA signaling in plants (Haudecoeur et al., 2009).

Neural Transporters
In neural tissues, two transporters of L-Pro are expressed; solute carrier family 6 member 7 (SLC6A7, PROT), a member of GABA family, and solute carrier family 6 member 19 (SLC6A19, B • AT1) FIGURE 5 | Proline is a neural metabotoxin. Proline is a metabolic precursor of L-glutamate and gamma-aminobutyric acid (GABA), i.e., the two major neurotransmitters in mammalian brain (top). At high plasma concentrations (2-3 millimolar instead of 150-200 micromolar), as occurs in patient suffering of hyperprolinemia type II (HPII , Table 3), the neurons can channeled free proline into glutamate biosynthesis, thus increasing free glutamate level. At a high level free proline can inhibit glutamate decarboxylase (GAD) enzyme (GABA biosynthesis) thus reducing GABA level in pre synaptic neurons. Altered levels of both these crucial neurotransmitters, and thus alterations in neurotransmission (middle), can explain some of the symptoms of hyperprolinemic patients, including schizophrenia. Defects in neural proline transport, which is mediated by different transporters such as the solute carrier family 6 member 7 (SLC6A7), a high affinity proline transporter, also known as proline transporter 1 (PROT1), and by the solute carrier family 6 member 19 (SLC6A19), also known as system B(0) neutral amino acid transporter 1 (B0AT1), are associated with ataxia and psychosis.

Neural Bioactive Peptides
Collagen-derived peptides such as Pro-Pro-OH induce the expression of crucial neural growth factors in the hippocampus of mice, increasing both dopamine concentration in the prefrontal cortex and proliferation of neural progenitor cells, and, eventually, reducing depression-like behavior (Mizushige et al., 2019;Nogimura et al., 2020). L-Pro-containing peptides (Gly-Pro-Glu and cyclo-Gly-Pro) inhibit inflammation and induce vascular remodeling, thereby protecting brain tissues from ischemic injury (Guan and Gluckman, 2009). Moreover, a phosphine analog of Pro-Gly-Pro tripeptide displays neuroprotective properties (Alexey et al., 2021).

Transforming Growth Factor
In ESCs, supplemental L-Pro induces expression of leftright determination factors (LEFTY1 and LEFTY2) and phosphorylation (activation) of small mother against decapentaplegic (SMAD2), which are extracellular inhibitors and intracellular effector of TGFβ-signaling, respectively (Figure 6; D' Aniello et al., 2015Aniello et al., , 2016. In VSMCs of injured arteries (Majesky et al., 1991;Ensenat et al., 2001), and in meniscal fibrochondrocytes (Pangborn and Athanasiou, 2005), supplemental TGFβ induces L-Pro uptake and collagen deposition. A L-Pro→TGFβ→L-Pro regulatory loop should allow the induction of collagen synthesis only when free L-Pro is sufficient to warrant timely tRNA loading, thus avoiding ribosome stalling (ER stress).

Wingless and Int-1
Pluripotent stem cells tend to proliferate as tightly packed cell aggregates, a trend that is inverted by a high L-Pro regimen (Comes et al., 2013). This phenotypic effect of L-Pro is fully counteracted by CHIR99021, a WNT signaling agonist. Moreover, L-Pro abundance delocalizes E-cadherin from the plasma membrane, where it is involved in cell-cell adherent junctions, to the Golgi. This subcellular redistribution of E-cadherin relies on the protein kinase domain containing, cytoplasmic (PKDCC), also known as vertebrate lonesome kinase (VLK) (Figure 6; Comes et al., 2013). L-Pro supplementation induces the expression of insulin-related genes such as IGF2, IGFR1, IGFBP3, IRS1 and IRS2 (D' Aniello et al., 2016), which are modulators of glycogen synthase kinase 3 (GSK3) activity (Desbois-Mouthon et al., 2001), and enhanced translation of collagen XVIII, which contains a frizzled-like domain (Heljasvaara et al., 2017), and can contribute to WNT modulation.

Reactive Oxygen Species and Hypoxia-Inducible Factors
Electrons released during mitochondrial L-Pro oxidation reduce flavin adenine dinucleotide (FAD) to generate FADH2 and/or O 2 during the production of ROS (Figure 6; Donald et al., 2001). In Arabidopsis thaliana, PRODH-mediated production of sub-lethal levels of ROS induces disease resistance (Cecchini et al., 2011), and in Caenorhabditis elegans this prolongs the nematodes life span (Zarse et al., 2012). In C. elegans, defects in L-Pro catabolism results in premature reproductive senescence and male infertility (Yen and Curran, 2021). In cancer cells, the L-Pro->PRODH->ROS axis can activate either pro-tumorigenic (cell survival) or anti-tumorigenic (cell death) signaling (Moloney and Cotter, 2018;Oscilowska et al., 2021). In rats' blood cells, hyperprolinemia increases oxidative damage of proteins, lipids and DNA (Ferreira et al., 2014). The effect of L-Pro on intracellular redox balance can be amplified by an NADPH-consuming futile cycle of L-Pro/P5C inter-conversion (Phang, 2019). Besides ROS, oxidative deamination of L-Pro generates α-KG, an essential substrate for hydroxylating dioxygenase enzymes, including PHD1-3 enzymes that catalyze the post-translational hydroxylation of specific proline residues of hypoxia-inducible factors (HIFs) resulting in destabilization of the protein. Indeed, the induction of PRODH activity in cancer cells destabilizes HIF1α and down-regulates the transcription of HIF1α target genes (Liu et al., 2009).

PROLINE IS AN EPIGENETIC MODIFIER
Several metabolites may influence, directly or indirectly, the activity of chromatin-modifying enzymes, and thus the epigenetic landscape of the cells (Reid et al., 2017;D'Aniello et al., 2019b;Surguchov et al., 2021). L-Pro is not a substrate, product, cofactor, or allosteric regulator of any epigenetic enzyme, but in ESCs its availability influences the activity of ten-eleven translocation (TET; DNA) and Jumonji (JMJ, histone) demethylase enzymes, which are strictly dependent on the availability of O 2 , α-KG, and ascorbic acid (vitamin C, VitC) to be active (Figure 7; Comes et al., 2013;D'Aniello et al., 2016D'Aniello et al., , 2019b.

DNA Methylation
L-Proline supplementation increases DNA 5-methylcytosine (5mC) and reduces 5-hydroxy-methylcytosine (5hmC) levels, inducing ∼1 × 10 3 DMRs distributed throughout all chromosomes of ESCs, with ∼50% of DMRs located in gene promoter regions (mostly H) and ∼20% in gene enhancers (D'Aniello et al., 2016). Importantly, ∼95% of genome sites hypermethylated after L-Pro supplementation are hypomethylated following VitC (50-150 µM) supplementation, FIGURE 7 | Proline is an epigenetic modifier. At a high proline regimen, extracellular proline is channeled into the cell cytoplasm through a transport system, as the solute carrier family 38 member 2 (SLC38A2), also known as system N amino acid transporter 2 (SNAT2), and used to charge tRNA molecules (top right), in a reaction catalyzed by the prolyl-tRNA synthetase (PRS). A high level of charged Proline-tRNA is an essential requisite for collagens synthesis (middle). A high fraction of L-Pro residues of the nascent molecules of collagens are hydroxylated by prolyl 4-hydroxylases (P4H 1, 2, 3) dioxygenases enzymes, a process that consume huge amounts of ascorbic acid (vitamin C, VitC) and α-ketoglutarate (α-KG) (middle right). VitC is transported by members of the solute carrier family 23 (SLC23A1, 2; bottom), whereas α-KG is produced inside mitochondria using proline and/or glutamate as precursors (top left). A sudden and sizeable increment of P4H activity in the endoplasmic reticulum (ER) can reduce the availability of VitC and α-KG for the activity of nuclear dioxygenases involved in DNA methylcytosine hydroxylation/demethylation (ten-eleven translocation, TET 1, 2, 3) and in histones lysine hydroxylation/demethylation (jumonji, JMJ) (bottom left). This compartmentalized metabolic perturbation, by increasing the DNA and histones methylation levels, can modify the epigenetic landscape of the cells.
indicating that L-Pro and VitC induce opposite epigenetic alterations in the same DNA regions. VitC is needed for the activity of TET demethylases (Blaschke et al., 2013), and ∼90% of genomic regions hypermethylated in by a high L-Pro regimen are hypermethylated also in cells lacking TET-mediated DNA demethylase activity (Lu et al., 2014;D'Aniello et al., 2019a).

Histone Methylation
L-Proline supplementation also triggers a genome-wide reprogramming of H3K9 methylation status, altering more than 1.6 × 10 4 genome sites located mainly in non-coding intergenic regions (Comes et al., 2013). Demethylation is catalyzed by members of the JMJ dioxygenase enzyme family, and upon silencing of Jmjd1a (H3K9 demethylase), ESCs adopt a molecular (upregulation of Fgf5 and Brachyury genes) and phenotypic (irregular flat-shaped colonies, sensitivity to trypsin digestion) state of pluripotency, similar to that induced by a high L-Pro regimen (Loh et al., 2007). Differences in the expression level and/or in the kinetic parameters (substrate affinity) of different JMJs can explain how L-Pro abundance alters the methylation level of some specific lysine residues (K9, K36) of histone H3.

Metabolic Imbalance
It recently emerged that a sudden and substantial increase in L-Pro stimulates collagen synthesis in the ER of ESCs (D'Aniello et al., 2019a), and that a significant fraction of L-Pro residues of nascent collagens are hydroxylated by prolyl 4-hydroxylase (P4H) dioxygenases, in particular by P4HA1 and P4HA2 enzymes, with depletion of α-KG and VitC. Under such conditions, nuclear dioxygenases such as TETs and JMJs lose activity, and consequently, DNA and histone methylation levels increase (Figure 7). Genetic and pharmacological evidence supports the idea that an abrupt induction of collagen synthesis leads to a

PROLINE INDUCES PROLIFERATION OF STEM AND TUMOR CELLS
Pluripotent stem cells shape the ICM in blastocysts of mammals and the apical meristems of plant organs (shoots and roots), and can self-renew and undergo differentiation into various somatic lineages. Cancer cells often display a stem cell-like growth behavior. Of note, L-Pro is a growth limiting metabolite (intrinsic starvation) for embryonic stem cells (D'Aniello et al., 2015), and for many different human cancer cells (D'Aniello et al., 2020). Similarly, L-Pro metabolism also influences the proliferation of meristematic and plant tumor cells (Trovato et al., 2001;Biancucci et al., 2015).
hypothesize that the induction of L-Pro accumulation during osmotic shock (see Figure 4), by altering the behavior/fate of stem cells, can contribute to couple a harmful environment (soil wetness) with the induction of organogenesis (root elongation).

Neoplastic Hairy Roots
L-Proline metabolism and plant tumor development are linked by the rolD gene of Agrobacterium rhizogenes, which encodes OCD that catalyzes L-Orn to L-Pro conversion, and is essential for the induction of neoplastic hairy roots (Figure 8; White et al., 1985;Costantino et al., 1994;Trovato et al., 2001). L-Pro accumulates in root tumor-like galls induced by the nematode Meloidogyne javanica or by Agrobacterium tumefaciens (Wachter et al., 2003;Trovato et al., 2018). Importantly, bacteria-induced tumorigenesis is attenuated in transgenic plants with low L-Pro levels (Haudecoeur et al., 2009).

Cytoskeletal Rearrangements
Embryonic stem cells seeded at a low density (50-250 cells/cm 2 ) in a high L-Pro regimen develop flat-shaped cell colonies formed by a core of adherent cells surrounded by a crown of detached cells showing mesenchymal features such as long actin stress fibers and mature focal adhesion complexes (Figure 9; Casalino et al., 2011;Comes et al., 2013). These L-Pro-induced cells are in a 'metastable' equilibrium, spread out from the colony core and rapidly moving back to re-establish adherent cell-cell contacts, a fully reversible phenotypic transition known as embryonic stem cell-to-mesenchymal transition (esMT) (Comes et al., 2013). Of note, in detached cells, E-cadherin is delocalized from the plasma membrane to the Golgi (see Figure 6) and unlike canonical EMT, during esMT the CDH1 gene is not down-regulated (Comes et al., 2013).

Invasion/Metastasis
After exposure to a high L-Pro regimen, ESCs acquire the ability to migrate through matrigel-coated porous membranes in response to serum gradients, or toward chemo-attractants such as EGF and stromal cell-derived factor 1 (Comes et al., 2013). These cells are able to reach the lung tissues after intravenous injection, and to generate tumors with a histological complexity of teratomas (Comes et al., 2013). Thus, a high L-Pro regimen converts adherent stem cells into spindle-shaped, motile and metastatic stem cells (Figure 9).

Metabolic Reprogramming
The morphological changes induced by L-Pro supplementation are associated with a metabolic switch from a bivalent to a more glycolytic metabolism. Indeed, metabolome profile analysis revealed higher lactate levels and increased susceptibility to 2-DG, a specific inhibitor of the glycolytic pathway (D'Aniello et al., 2016). Moreover, a high L-Pro regimen reduces the mitochondrial membrane potential, which relies on oxidative phosphorylation rates (D'Aniello et al., 2017), thus supporting glycolytic energy metabolism.
Pluripotency L-Proline supplementation remodels the transcriptome of naïve ESCs by altering the expression of ∼1.5 × 10 3 protein-coding genes mainly related to cell adhesion, cell junction, and cell motility functions (Comes et al., 2013;D'Aniello et al., 2017). Cells treated with L-Pro are leukemia inhibitory factor (LIF)dependent, express pluripotency markers as Nanog homeobox, can differentiate into cardiomyocytes and neurons in vitro, and are able to colonize mouse blastocysts (chimeric embryos; Figure 9; Casalino et al., 2011). Recently, Cermola et al. (2021) reported that L-Pro-treated ESCs can differentiate into primordial germ cell like cells (PGCLCs), and are competent to develop elongated gastruloids, suggesting that L-Pro abundance drives ESCs into an early primed state of pluripotency.

CONCLUSION AND PERSPECTIVES
The control of L-Pro metabolism in human cells is relatively poorly understood, even though it might have a great impact on human health (Figure 10). For instance, PrAMPs displaying potent antimicrobial activity and low toxicity for human cells could be efficient tools to fight multidrug-resistant pathogens, a serious public health concern (Charon et al., 2019). Salivary proline-rich peptides able to neutralize microbe attacks could contribute to avoiding the development of dental caries, an infectious disease that affects billions of people (Werneck et al., 2010;Stromberg et al., 2017). Moreover, salivary proteins could contribute to food choices, and so to nutrition status and health (Melis et al., 2021). Translational suppression of proline-rich proteins by pharmacological targeting of the PRS is emerging as an attractive therapeutic approach for the treatment of different diseases. Of note, halofuginone, a specific inhibitor of the PRS, is already in clinical trials for the treatment of fibrotic diseases (Pines and Spector, 2015), and has been recently shown to inhibit SARS-CoV-2 infection, suppressing the translation of proline-rich host attachment factors (Sandoval et al., 2021). Exploitation of L-Pro as a source of carbon and/or energy appears to be an adaptive response of cells to high-L-Pro microenvironments, which can be generated by pathological tissue damage (bacterial invasion, cancer progression, trauma). Although never measured, it is possible to speculate that in an extremely confined extracellular space, free L-Pro can reach exceptionally high concentrations. L-Pro supports invasiveness of bacteria, parasites and cancer cells, all processes that engage tissue degradation/remodeling (Christgen and Becker, 2019;D'Aniello et al., 2020), and D-Pro-derived peptidomimetic inhibitors of human gelatinases/metalloproteinases involved in tissue remodeling are potential anti-metastatic agents (Lenci et al., 2021). Moreover, enzymes involved in L-Pro metabolism are potential targets of antiparasitic drugs (Saye et al., 2017;Ugwu et al., 2018).
Various stressful conditions, including suboptimal temperature, high salinity and oxidative agents, can alter the conformations of proteins and other macromolecules. Since L-Pro is a potent and non-toxic chemical chaperone, its intracellular accumulation could be an evolutionarily conserved response aimed at inhibiting the formation of unfolded/misfolded protein aggregates. Indeed, hemocompatible gold nanoparticles coated with L-Pro inhibit both collagen fibril formation (Anand et al., 2017) and insulin aggregation (Prajapati et al., 2021), and could provide a basis for creating antifibrotic and antiamyloid formulations.
Numerous studies conclude that at high levels, free L-Pro is a neurotoxin. Lactic acid inhibits PRODH activity, and lactic acidosis syndrome (blood lactic acid >5 mM) is frequently associated with hyperprolinemia, supporting the idea that in adult humans L-Pro homeostasis is strictly dependent on L-Pro oxidation. Of note, L-Pro at high levels is harmful for brain/neural activity, but acting as a chemical chaperone it can prevent protein unfolding/misfolding (Liang et al., 2014). Thus, regulation of L-Pro metabolism is studied in the context of neurodegenerative diseases associated with the formation of protein aggregates, as exemplified by Huntington's, Parkinson's, and Alzheimer's (Powers et al., 2009;Khan et al., 2010).
Beyond some cancer cells, whether and which normal human cells oxidize L-Pro, and whether this contributes to maintain prolinemia, remains unknown. The concomitant activation of L-Pro oxidation (for ATP production in mitochondria) and tRNA loading (for collagen synthesis in the ER) remains uncharacterized at the single-cell level. By generating sublethal amounts of ROS, L-Pro oxidation can induce redox signaling, and eventually a compensatory stress response, through the induction of ROS consuming/neutralizing enzymes. Importantly, in bacteria (Zhang et al., 2015), fungi (Chen and Dickman, 2005) and nematodes (Zarse et al., 2012), L-Pro oxidation increases cell resilience to stressful conditions. However, the induction of stress tolerance by L-Pro oxidation in human cells remains an open question.
Aging is usually associated with a significant reduction (quantitative and qualitative) in CTs (tendon, bone, cartilage), for which L-Pro is essential. Of note, older people and patients suffering hereditary defects L-Pro biosynthesis share a similar aged appearance (e.g., osteopenia, cataracts, wrinkled skin, cutis laxa). Furthermore, sedentary life-induced sarcopenia is associated with hyperprolinemia, but its impact on neural disorders suffered by the elderly is unknown.
How L-Pro availability modulates stem and cancer cell proliferation is an interesting question that is getting increasingly attention. Free L-Pro can improve the translation of L-Prorich proteins (Sabi and Tuller, 2015;Chyzynska et al., 2021) or simple protein stretches, as demonstrated for HOXB4 involved in leukemia (Cusan et al., 2017). Recently, cell-based drug screening identified 137 drugs (out of 1200 assayed) able to inhibit stem cell proliferation, of which 80% also inhibited cancer cells (D'Aniello et al., 2019a), suggesting a similar chemosensitivity spectrum.
Thus, the development of therapeutic strategies to target L-Pro metabolism may provide new options to eradicate cancer cells. Importantly, L-Pro abundance induces invasiveness in stem cells, a peculiar trait of migrating cancer cells. Certainly, the ability of L-Pro to control morphogenesis is not limited to stem cells. For instance, L-Pro availability influences plant shoot and root development (see Biancucci et al., 2015, for a review), hyphal morphology in the pathogenic fungus Colletotrichum trifolii (Memmott et al., 2002), and filamentation (yeast-tohyphal transition) in the pathogenic yeast Candida albicans (Dabrowa et al., 1976;Silao et al., 2019).

AUTHOR CONTRIBUTIONS
EP and GM contributed to the conception and design of the review. FC, CD'A, AF, OG, and DD performed the literature search, and wrote the first draft of the manuscript. EP and FC prepared the figures. EP and GM critically revised the text and provided substantial scientific contribution. All authors approved the final version of the manuscript.