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).

L-Proline-rich antimicrobial peptides (PrAMPs), involved in innate immunity, are the first line of defense against infections (Graf and Wilson, 2019), and they contain up to 50% L-Pro residues, and are secreted by insects, crustaceans and mammals (Table 3; Mishra et al., 2018). Mechanistically, PrAMPs are channeled by the peptide antibiotic transporter SbmA into the bacterial cytoplasm (Mattiuzzo et al., 2007; Runti et al., 2013), where they bind ribosomal proteins and inhibit protein synthesis (Figure 2 and Table 3; Graf et al., 2017; Graf and Wilson, 2019; Baliga et al., 2021).

Unstructured L-Pro-rich salivary proteins (PRPs) contain up to 40% L-Pro residues and account for ∼70% of total proteins in human saliva (Messana et al., 2015; Lorenzo-Pouso et al., 2018). The acinar cells of parotid and submandibular salivary glands synthesize and secrete acidic (aPRP) and basic (bPRP) proteins (Figure 2). While aPRPs bind calcium and protect the tooth surface, bPRPs bind polyphenols/tannins inducing the astringency sensation that influences diet selection (Canon et al., 2021; Dufourc, 2021). Since tannins induce ER stress and ATF4 expression (Nagesh et al., 2018), and since ATF4 in turn induces the transcription of L-Pro biosynthesis genes (ALDH18A1 and PYCR1) (Han et al., 2013; D’Aniello et al., 2015; Gonen et al., 2019), it is tempting to hypothesize that a neutralizing response axis (ER stress→ATF4→L-Pro biosynthesis→PRP synthesis/secretion) can be induced by tannins in salivary glands.

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).

The extracellular space in plants and algae contains up to 10% dry weight of hydroxyproline (L-Pro-OH)-rich glycoproteins (HRGPs) such as extensins (Showalter, 1993; Lamport et al., 2011), in which L-Pro-OH constitutes up to 30% of total amino acids (Kieliszewski and Lamport, 1994). Besides being structural pilasters, HRGPs are involved in (i) tissue/organ development (embryo, xylem, pod, root hairs, pollen) (Wu et al., 2001; Velasquez et al., 2011; Ogawa-Ohnishi et al., 2013), (ii) a defense mechanism against environmental stress (heat stress, mechanical wounding and bacterial infection) (Francisco and Tierney, 1990; Zhang et al., 2021b), and (iii) an oxygen barrier in the parenchyma of nitrogen-fixing legume root nodules (nodulins) (Scheres et al., 1990; Sherrier et al., 2005). HRGP synthesis requires free L-Pro, and plants respond to pathogen attack by inducing L-Pro accumulation and HRGP synthesis (Fabro et al., 2004).

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^V12) 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).

Protozoan parasites adapt their metabolism to the mutable environments encountered throughout their life cycle, including the hemolymph of their insect vectors (Bringaud et al., 2012). Trypanosoma brucei, the causative agent of sleeping sickness, is transmitted by tsetse flies (Glossina diptera), and both organisms can oxidize L-Pro to accomplish ATP biosynthesis (Figure 3; Michalkova et al., 2014; Mantilla et al., 2017; Smith et al., 2017; Dolezelova et al., 2020; Haindrich et al., 2021; Villafraz et al., 2021). L-Pro sustains Trypanosoma cruzi (the causative agent of Chagas disease) cell invasion and intracellular epimastigote-to-trypomastigote transition (Figure 3; Martins et al., 2009; Mantilla et al., 2015; Barison et al., 2017). Parasites also utilize L-Pro for anabolic purposes. For instance, halofuginone, a selective inhibitor of PRS, blocks the synthesis of L-Pro-rich proteins and the proliferation of Plasmodium falciparum (the causative agent of malaria) (Hewitt et al., 2017).

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).

Some cells use the carbon skeleton of L-Pro to synthesize L-ornithine and L-arginine. For instance, in the gut of neonates, L-glutamate to pyrroline-5-carboxylate conversion is negligible, 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., 2005, 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).

Hypertonic shocks induce water outflow, which reduces the cell volume and lowers macromolecule stability (Burg et al., 2007; Hoffmann et al., 2009; Stadmiller et al., 2017). Cells respond by accumulating L-Pro, which generates an opposite force of water retention (Figure 4). In bacteria, L-Pro accumulation occurs by uptake of extracellular free L-Pro after the induction (up to 700-fold) of a low-affinity L-Pro transporter (Csonka and Hanson, 1991), through degradation of extracellular L-Pro-rich proteins (Zaprasis et al., 2013) and/or de novo L-Pro biosynthesis (Patel et al., 2018). The ability to accumulate L-Pro is vital to organisms inhabiting mutable (fresh/brackish water, intertidal) habitats, such as gastropod mollusks (Wiesenthal et al., 2019). Plants respond to drought, salinity and freezing temperatures by accumulating L-Pro (Yoshiba et al., 1997; Szabados and Savoure, 2010; Hnilickova et al., 2021; Papu et al., 2021), and in tomato cells concentrations can reach 60 mM (500-fold higher than normal levels) (Handa et al., 1983). L-Pro accumulation protects human cells from hyperosmotic stress (Thiemicke and Neuert, 2021). Indeed, L-Pro uptake facilitates the recovery a viable cell volume after hypertonic stress (Law, 1991; Bevilacqua et al., 2005; Krokowski et al., 2017), and the PP1 phosphatase subunit protein PPP1R15A/GADD34 promotes cis-to-trans Golgi trafficking, and the plasma membrane localization of SLC38A2 L-Pro transporter (Figure 4; Krokowski et al., 2017).

In yeast, L-Pro accumulation confers ethanol and freezing tolerance (Takagi, 2008). In overwintering insects, L-Pro contributes to water retention and freezing tolerance (Figure 4), and levels increase to ∼80% of the total pool of free amino acids (Kostal et al., 2011, 2016; Rozsypal et al., 2018; Stetina et al., 2018). Of note, hyperprolinemic larvae of the fly Chymomyza costata can survive immersion in liquid nitrogen (−196°C) (Kostal et al., 2011). In Drosophila larvae, an L-Pro-rich diet increases the whole-body L-Pro concentration (up to 60 mM) and freezing tolerance (Kostal et al., 2012).

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 contributes to protect plants from photo-oxidative stress (i.e., light-dependent generation of ROS) (Liang et al., 2013). Recently, it emerged that salivary L-Pro-rich proteins can neutralize ROS, and specifically hydroxyl radicals (Komatsu et al., 2020).

In plants, L-Pro is accumulated after exposure to heavy metals such as cadmium, chromium, and zinc (Sharma et al., 1998; Verbruggen and Hermans, 2008; Hayat et al., 2012; Dubey et al., 2018; Dong et al., 2021; Pejam et al., 2021; Zdunek-Zastocka et al., 2021), and this mitigates the detrimental effects of cadmium in young olive plants (Zouari et al., 2016) and cultured tobacco cells (Islam et al., 2009). Heavy metal toxicity is usually associated with ROS accumulation (Figure 4). Indeed, cadmium induces p53 (Aimola et al., 2012), a transcriptional inducer of PRODH expression (Polyak et al., 1997), which catalyzes L-Pro oxidation in mitochondria, leading to abnormal ROS production and apoptosis (Liu et al., 2006, 2008, 2009; Oscilowska et al., 2021). Thus, a p53→PRODH→ROS→apoptosis axis may be activated as a response to toxic metals such as cadmium.

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 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.

Hyperprolinemia is an etiopathogenetic factor of schizophrenia, a heterogeneous disorder that affects about 21 million people worldwide (Disease et al., 2017). HPI Drosophila models (PRODH mutants) exhibit a depressed ‘sluggish’ behavior (Hayward et al., 1993), while HPII models (defects in P5C to L-glutamate conversion due to a P5CDH mutation) display larval and pupal lethality (He and DiMario, 2011). Conversely, PRODH-overexpressing flies exhibit an opposite ‘aggressive’ behavior (Zwarts et al., 2017). HPI mouse models also exhibit sluggish movements (Blake and Russell, 1972; Kanwar et al., 1975) and schizophrenia-related phenotypes (learning, memory and sensorimotor gating) (Gogos et al., 1999; Paterlini et al., 2005). 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 L-glutamate, 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).

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; Roigaard-Petersen and Sheikh, 1984; Malandro and Kilberg, 1996; Thwaites and Anderson, 2007, 2011; Verrey et al., 2009). Genetic and/or pharmacological inhibition of SLC6A7 reduces locomotor activity and improves mouse learning and memory (Zipp et al., 2014; Schulz et al., 2018). SLC6A7 is induced in fibroblasts of patients suffering of Friedreich’s ataxia, characterized by a lack of control in muscle activity/movements (Napierala et al., 2017). Mutations of SLC6A19 are associated with Hartnup disease, a complex syndrome involving cerebellar ataxia and psychosis (Seow et al., 2004). SLC6A20 (IMINO) is expressed in human neurons and regulates L-Pro and glycine homeostasis (Bae et al., 2021).

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).

Genetic defects in PYCR2, a PYCR1 paralog, are associated with leukodystrophy-hypomyelinating 10 (HLD10; Table 2), a syndrome characterized by microcephaly and psychomotor disability (Nakayama et al., 2015; Zaki et al., 2016). PYCR2-deficient fibroblasts derived from HLD10 patients are highly susceptible to oxidative stress-induced apoptosis, and this may contribute to this complex phenotype (Reversade et al., 2009; Nakayama et al., 2015).

In cultured ESCs, exogenously available L-Pro, at a physiological concentration range (50–250 μM), disables the AAR pathway by improving L-Pro-tRNA loading, inactivating (dephosphorylation) eukaryotic translation initiation factor alpha (EIF2α), and eventually, preventing translation of ATF4 mRNA (Figure 6; D’Aniello et al., 2015). In the absence of ATF4, the genes involved in L-Pro biosynthesis (ALDH18A1 and PYCR1), and L-Pro uptake (SLC38A2 and GADD34) are silenced (Gaccioli et al., 2006; D’Aniello et al., 2015). L-Pro-ATF4 interplay also impacts cardiac fibroblast metabolism (Qin et al., 2017). Human kidney and breast cancer cells suffer from a similar intrinsic and partial shortage of L-Pro (Loayza-Puch et al., 2016; Sahu et al., 2016).

In stem and cancer cells, a high L-Pro regimen induces phosphorylation of ERK1 and enhances the transcription of ERK-related genes (Figure 6; Liu et al., 2006; D’Aniello et al., 2016). Supplemental L-Pro induces the expression of growth factors (FGF5, FGF8, and FGF13) and the synthesis of collagen, and this can contribute to the induction of the ILK/ERK super-pathway, as revealed by transcriptome analysis (Comes et al., 2013; D’Aniello et al., 2016, 2019b). Indeed, collagen mimics consisting of repeated units (5 or 10) of the Pro-Pro-Gly tripeptide activate phosphoinositide 3-kinase (PI3K)-dependent p38 mitogen-activated protein kinase (MAPK) phosphorylation (Weinberger et al., 2005).

In ESCs, supplemental L-Pro induces expression of left-right 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., 2015, 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).

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.

In mouse ESCs, L-Pro supplementation enhances L-Pro-tRNA loading and inhibits autophagy. Accordingly, halofuginone inhibits L-Pro-tRNA loading and activates autophagy (D’Aniello et al., 2015). In human and murine ECSLC, knockdown of Tap73 tumor protein reduces L-Pro biosynthesis and induces autophagy (Sharif et al., 2019). Protracted exposure to free L-Pro induces stem cell motility, invasiveness, and macro-autophagy (D’Aniello et al., 2015). In cancer cells overexpressing PRODH and exposed to a high exogenous L-Pro regimen, autophagy is induced (Liu et al., 2012b).

Electrons released during mitochondrial L-Pro oxidation reduce flavin adenine dinucleotide (FAD) to generate FADH2 and/or O₂ 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).

L-Proline supplementation increases DNA 5-methylcytosine (5mC) and reduces 5-hydroxy-methylcytosine (5hmC) levels, inducing ∼1 × 10³ 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, 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).

L-Proline supplementation also triggers a genome-wide reprogramming of H3K9 methylation status, altering more than 1.6 × 10⁴ 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.

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 similar metabolic imbalance and epigenome alterations also in cancer cells (D’Aniello et al., 2019a).

Supplemental L-Pro (50–250 μM) improves proliferation of ESCs (Figure 8; Washington et al., 2010; Casalino et al., 2011), development of pre-implantation embryos (Morris et al., 2020) and fetus survival (Liu et al., 2019). L-Pro is internalized into stem cell cytoplasm through the SLC38A2 (SNAT2) transporter (Tan et al., 2011), and halofuginone (prolyl-tRNA synthetase inhibitor) fully counteracts L-Pro induction of cell proliferation (D’Aniello et al., 2015). Moreover, halofuginone and L-Pro modify the ESC transcriptome in opposite directions (D’Aniello et al., 2015), showing that mouse ESCs are partially starved of L-Pro, even after incubation in complete rich medium. Of note, during in vitro fertilization of mouse oocytes, L-Pro supplementation improves stem cells (ICM) proliferation and embryo development (Treleaven et al., 2021).

L-Proline shortage is a major cause of partial ribosome stalling (diricore analysis) suffered by kidney and breast cancer cells (Loayza-Puch et al., 2016). Likewise, up-regulation of L-Pro biosynthesis genes (ALDH18A1 and PYCR1) also reveals L-Pro starvation in tumor cells (D’Aniello et al., 2020). Moreover, ALDH18A1 knock-down activates AAR stress signaling, and reduces melanoma tumor growth both in vitro and in vivo (Kardos et al., 2015), whereas PYCR1 induction improves proliferation and invasiveness of breast, esophagus, lung, melanoma, pancreas, and prostate cancer cells (Nilsson et al., 2014; Ding et al., 2017; Zeng et al., 2017; Cai et al., 2018; Ye et al., 2018; Kardos et al., 2020; Forlani et al., 2021). Of note, kindlerin 2 (KINDLING-2) protein stabilizes the mitochondrial PYCR1 enzyme, increasing L-Pro synthesis and lung adenocarcinoma cell proliferation (Guo et al., 2019). Importantly, translocation of KINDLING-2 into mitochondria is regulated by ECM stiffness (Guo et al., 2019) and PINCH-1 (particularly interesting new Cys-His protein 1) protein (Guo et al., 2020; Ding et al., 2021), and PYCR1 activity is modulated by the mitochondrial deacetylase sirtuin (SIRT3) (Chen et al., 2019). PYCR1 stabilization by KINDLING-2 induces L-Pro synthesis in human lung fibroblasts and contributes to pulmonary fibrosis progression (Zhang et al., 2021a).

Post-embryonic organogenesis in adult plants relies on apical meristems, and a fine-tuned balance between self-renewal and differentiation fates adapts organ morphogenesis to a fluctuating environment (Figure 8; Mattioli et al., 2009; Lehmann et al., 2010; Szabados and Savoure, 2010). In Arabidopsis, L-Pro availability controls root meristem activity (Biancucci et al., 2015) by modifying the expression of L-Pro-rich proteins, and regulating a compartmentalized (mitochondria/cytoplasm) cycle of L-Pro synthesis and degradation that modifies the NADP⁺/NADPH ratio (Verslues and Sharma, 2010). Therefore, it is tempting to 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).

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).

Embryonic stem cells seeded at a low density (50–250 cells/cm²) 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).

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).

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.

L-Proline supplementation remodels the transcriptome of naïve ESCs by altering the expression of ∼1.5 × 10³ 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.

L-Proline-induced esMT is inhibited by well-known chemical modulators of key signaling pathways such as CHIR99021 (WNT agonist) and PD0325901 (TGFβ antagonist) (D’Aniello et al., 2016). Moreover, D’Aniello et al. (2019a) made use of the cell colony morphology to develop a high-throughput screening method, and identified 14 FDA-approved drugs (from 1200 assayed) able to inhibit esMT without preventing L-Pro-induced cell proliferation. Spiramycin (macrolide), Propafenone (flavonoid) and Budesonide (steroid) inhibit esMT and have very different chemical structures, implying molecular complexity in L-Pro-mediated control of stem cell plasticity. Importantly, VitC, but not other antioxidants such as NAC, is a full inhibitor of esMT (D’Aniello et al., 2016).