key: cord-0067016-2qotakpz
authors: Patriarca, Eduardo J.; Cermola, Federica; D’Aniello, Cristina; Fico, Annalisa; Guardiola, Ombretta; De Cesare, Dario; Minchiotti, Gabriella
title: The Multifaceted Roles of Proline in Cell Behavior
date: 2021-08-12
journal: Front Cell Dev Biol
DOI: 10.3389/fcell.2021.728576
sha: 1117b8ea56c3bc9fa197d682e3148f0e6918e49a
doc_id: 67016
cord_uid: 2qotakpz

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.

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 ).

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) .

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) 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 FIGURE 1 | Proline structure, uses, biosynthesis and degradation/oxidation. Chirality of pyrrolidine-2-carboxylic acid (top), known as proline (CAS: 147-85-3, EC: 205-702-2, CHEBI: 17203, HMDB0000162, MW = 115.13 g/mol). Of the two enantiomers (L and D) living cells metabolize predominantly the L-proline enantiomer (top). Proline is an organocatalyst used to synthesize enantiopure drugs (middle top). Proline is also a potent chemical chaperone able to stabilize proteins in their natural conformation and thus, it is used to cryopreserve living cells/organisms (middle bottom). Due to its pyrrolidine ring structure, the enzymes involved in de novo L-proline biosynthesis, namely aldehyde dehydrogenase 18 family member A1 (ALDH18A1) and pyrroline-5-carboxylate reductase 1 (PYCR1), as well as the enzymes involved in L-proline oxidation, namely proline dehydrogenase (PRODH) and the pyrroline-5-carboxylate dehydrogenase (P5CDH), are highly specific (bottom).

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) .

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.

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) .

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 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., 2005 Wu 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) .

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).

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 (Kostal et al., , 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 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).

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 (Liu et al., , 2008 (Liu et al., , 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 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 .

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, PRODHoverexpressing 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 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 ).

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.

( Figure 5 ; Roigaard-Petersen and Sheikh, 1984; Malandro and Kilberg, 1996; 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) . PYCR2deficient 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) .

The availability of some amino acids influences the activity of cell signaling pathways. For instance, the level of L-glutamine, L-leucine, and L-arginine impacts the mechanistic target of rapamycin (mTOR) pathway (Curi et al., 2007; Xie and Klionsky, 2007; Ryter et al., 2013; Bar-Peled and Sabatini, 2014; Lahiri et al., 2019) . L-tyrosine and L-phenylalanine modulate the G protein-coupled receptor 142 (GPR142)-mediated pathway (Lin et al., 2016) . It emerged that mESCs, isolated from mouse blastocysts, suffer from a finely regulated partial shortage of L-Pro, and that an increase in free L-Pro availability modulates the activity of the amino acid stress response (AAR), fibroblast growth factor/extracellular signal-related kinase (FGF/ERK), TGFβ, wingless and int-1 (WNT), and redox signaling pathways. As expected, specific signaling modulators such as halofuginone (AAR inducer), SB431542 (TGFβ inhibitor), CHIR99021 (WNT agonist) and PD0325901 (MEK/ERK inhibitor) fully counteract L-Pro supplementation effects (Comes et al., 2013; D'Aniello et al., 2015) . Moreover, L-Pro impacts mTOR pathway in porcine trophectoderm cells .

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 ERKrelated 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 superpathway, as revealed by transcriptome analysis (Comes et al., 2013; D'Aniello et al., 2016 D'Aniello et al., , 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 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., 2015 Aniello 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).

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 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 ).

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., 2016 D'Aniello et al., , 2019b .

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) .

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.

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 

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) .

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 . 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) . 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 FIGURE 10 | This illustration encompasses the multifaceted roles of proline in cell biology, including: building block of proteins (collagens, cornifins, salivary proteins); energy fuel (bacteria, parasites, cancer cells); stress defender (cold shock, drought/salinity, ER stress); proliferation inducer (stem, meristematic, cancer cells); plasticity controller (cell shape, motility); epigenetic modifier (DNA/histone methylation); modulator of cell signaling pathways (AAR, autophagy, ERK, TGFβ) and neural toxin associated (schizophrenia).

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 ).

Some metabolites modulate relevant phenotypic transformations such as stem cell differentiation, somatic cell reprogramming, and EMT. For instance, butyric acid drives the differentiation of MSCs into adipocytes (Tugnoli et al., 2019) , and, conversely, enhances the reprogramming efficiency of fetal fibroblasts into pluripotent cells (Liang et al., 2010; Mali et al., 2010) . Likewise, VitC improves cell differentiation (Cao et al., 2012) and reprogramming (Esteban et al., 2010) . Similarly, L-Pro governs the morphology, migratory behavior and pluripotency state of stem cells (Washington et al., 2010; Casalino et al., 2011) .

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) .

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.

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. 

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) .

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. 

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Physiology of cell volume regulation in vertebrates

Improving viscosity and stability of a highly concentrated monoclonal antibody solution with concentrated proline

Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant

Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells

The severe form of type I hyperprolinaemia results from homozygous inactivation of the PRODH gene

Hyperprolinemia is a risk factor for schizoaffective disorder

PRODH mutations and hyperprolinemia in a subset of schizophrenic patients

Plasma proline kinetics and concentrations in young men in response to dietary proline deprivation

Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure

New lethal disease involving type I and III collagen defect resembling geroderma osteodysplastica, De Barsy syndrome, and Ehlers-Danlos syndrome IV

Biochemical, morphological and hybrid studies in hyperprolinemic mice

Schizophrenia susceptibility associated with interstitial deletions of chromosome 22q11

Salubrinal in combination with 4E1RCat synergistically impairs melanoma development by disrupting the protein synthetic machinery

Disruption of proline synthesis in melanoma inhibits protein production mediated by the GCN2 pathway

Metabolic epilepsy in hyperprolinemia type II due to a novel nonsense ALDH4A1 gene variant

Naturally occurring organic osmolytes: from cell physiology to disease prevention

Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri, the vector of huanglongbing

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder

Oncocin (VDKPPYLPRPRPPRRIYNR-NH2): a novel antibacterial peptide optimized against gram-negative human pathogens

Direct evaluation of the antioxidant properties of salivary proline-rich proteins

Arginine and proline applied as food additives stimulate high freeze tolerance in larvae of Drosophila melanogaster

Conversion of the chill susceptible fruit fly larva (Drosophila melanogaster) to a freeze tolerant organism

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen

Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin

Defect in proline synthesis: pyrroline-5-carboxylate reductase 1 deficiency leads to a complex clinical phenotype with collagen and elastin abnormalities

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic

Short Proline-Rich antimicrobial peptides inhibit either the bacterial 70S ribosome or the assembly of its large 50S subunit

GADD34 function in protein trafficking promotes adaptation to hyperosmotic stress in human corneal cells

Watch what you (Self-) eat: autophagic mechanisms that modulate metabolism

Role of the extensin superfamily in primary cell wall architecture

Amino acids as volume-regulatory osmolytes in mammalian cells

Proline metabolism and transport in plant development

Multitargeting application of proline-derived peptidomimetics addressing cancer-related human matrix metalloproteinase 9 and carbonic anhydrase II

Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties

A new method for isolation of hydroxy-L-proline and L-proline from gelatin

L-Proline: an effective agent for frozen and post-thawed donkey semen storage

Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth

Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action

Butyrate promotes induced pluripotent stem cell generation

Proline biosynthesis is required for endoplasmic reticulum stress tolerance in Saccharomyces cerevisiae

Proline mechanisms of stress survival

Compound heterozygous mutations in PYCR1 further expand the phenotypic spectrum of De Barsy syndrome

GPR142 controls tryptophan-induced insulin and incretin hormone secretion to improve glucose metabolism

In vitro activity of PR-39, a proline-arginine-rich peptide, against susceptible and multi-drugresistant Mycobacterium tuberculosis

Immunohistochemical expression of extracellular matrix proteins and adhesion molecules in pancreatic carcinoma

A novel proline-catalyzed three-component reaction of ketones, aldehydes, and Meldrum's acid. Synlett 1687-1689

New mechanistic studies on the proline-catalyzed aldol reaction

Genetic variation at the 22q11 PRODH2/DGCR6 locus presents an unusual pattern and increases susceptibility to schizophrenia

Maternal L-proline supplementation enhances fetal survival, placental development, and nutrient transport in micedagger

L-Proline activates mammalian target of rapamycin complex 1 and modulates redox environment in porcine trophectoderm cells

Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments

Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC

Proline oxidase functions as a mitochondrial tumor suppressor in human cancers

Proline oxidase activates both intrinsic and extrinsic pathways for apoptosis: the role of ROS/superoxides, NFAT and MEK/ERK signaling

Proline oxidase, a p53-induced gene, targets COX-2/PGE2 signaling to induce apoptosis and inhibit tumor growth in colorectal cancers

Tumour-specific proline vulnerability uncovered by differential ribosome codon reading

Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells

Protein-Based salivary profiles as novel biomarkers for oral diseases

Role of tet proteins in enhancer activity and telomere elongation

Identification of candidate biomarkers and pathways associated with psoriasis using bioinformatics analysis

Isolation from an ant Myrmecia gulosa of two inducible O-glycosylated proline-rich antibacterial peptides

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9

Production of transforming growth factor beta 1 during repair of arterial injury

Molecular biology of mammalian amino acid transporters

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes

Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution

Proline metabolism is essential for trypanosoma Brucei brucei survival in the tsetse vector

Role of Delta1-pyrroline-5-carboxylate dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi

Fragments of the non-lytic proline-rich antimicrobial peptide Bac5 kill E. coli cells by inhibiting protein synthesis

The dolphin proline-rich antimicrobial peptide Tur1A inhibits protein synthesis by targeting the bacterial ribosome

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis

Use of L-proline and ATP production by Trypanosoma cruzi metacyclic forms as requirements for host cell invasion

Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids

The ambivalent role of proline residues in an intrinsically disordered protein: from disorder promoters to compaction facilitators

In vivo target exploration of apidaecin based on acquired resistance induced by gene overexpression (ARGO assay)

Proline accumulation in plants: not only stress

Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides

Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase

Differences in salivary proteins as a function of PROP taster status and gender in normal weight and obese subjects

Proline reverses the abnormal phenotypes of Colletotrichum trifolii associated with expression of endogenous constitutively active Ras

Chrono-proteomics of human saliva: variations of the salivary proteome during human development

Vitamin B6 generated by obligate symbionts is critical for maintaining proline homeostasis and fecundity in tsetse flies

Tryptophan-Rich and proline-rich antimicrobial peptides

Ginger-Degraded Collagen hydrolysate exhibits antidepressant activity in mice

ROS signalling in the biology of cancer

Proline: the distribution, frequency, positioning, and common functional roles of proline and polyproline sequences in the human proteome

Selected amino acids promote mouse pre-implantation embryo development in a growth factor-like manner

Targeting polyamine metabolism for cancer therapy and prevention

Toxicity of L-proline toward rat hippocampal neurons

ALDH4A1 expression levels are elevated in postmortem brains of patients with schizophrenia and are associated with genetic variants in enzymes related to proline metabolism

Tannic acid induces endoplasmic reticulum stressmediated apoptosis in Prostate cancer

Mutations in PYCR2, encoding Pyrroline-5-Carboxylate reductase 2, cause microcephaly and hypomyelination

Efficacy of proline in the treatment of menopause

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

Proline dehydrogenase is essential for proline protection against hydrogen peroxide-induced cell death. Free Radic

Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

Putative mitochondrial alphaketoglutarate-dependent dioxygenase Fmp12 controls utilization of proline as an energy source in Saccharomyces cerevisiae

Prolyl-hydroxyproline, a collagen-derived dipeptide, enhances hippocampal cell proliferation, which leads to antidepressant-like effects in mice

Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana

Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions

Proline oxidase silencing inhibits p53-dependent apoptosis in MCF-7 breast cancer cells

Effects of growth factors on meniscal fibrochondrocytes

ALDH18A1 gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism

Physiological, biochemical, and anatomical responses of Araucaria araucana seedlings to controlled water restriction

Disease variants of human Delta(1)-pyrroline-5-carboxylate reductase 2 (PYCR2)

Halotolerant Exiguobacterium profundum PHM11 tolerate salinity by accumulating L-Proline and fine-tuning gene expression profiles of related metabolic pathways

Transcriptional and behavioral interaction between 22q11.2 orthologs modulates schizophrenia-related phenotypes in mice

Zinc oxide nanoparticles mediated substantial physiological and molecular changes in tomato

Re-optimization of the organocatalyzed double aldol domino process to a key enal intermediate and its application to the total synthesis of Delta(1)(2)-Prostaglandin J(3)

Proline: mother Nature's cryoprotectant applied to protein crystallography

Proline metabolism in cell regulation and cancer biology: recent advances and hypotheses

Halofuginone -the multifaceted molecule

A model for p53-induced apoptosis

Biological and chemical approaches to diseases of proteostasis deficiency

Osmoprotectant coated thermostable gold nanoparticles efficiently restrict temperature-induced amyloid aggregation of insulin

The human serum metabolome

Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics

Effects of natural deep eutectic solvents on lactic acid bacteria viability during cryopreservation

Activation of the amino acid response pathway blunts the effects of cardiac stress

Bioinspired l-Proline oligomers for the cryopreservation of oocytes via controlling ice growth

The effects of interactions between proline and carbon nanostructures on organocatalysis in the Hajos-Parrish-Eder-Sauer-Wiechert reaction

Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome

The impact of cellular metabolism on chromatin dynamics and epigenetics

Mutations in PYCR1 cause cutis laxa with progeroid features

Renal transport of neutral amino acids. Demonstration of Na+-independent and Na+-dependent electrogenic uptake of L-proline, hydroxy-L-proline and 5-oxo-L-proline by luminal-membrane vesicles

Identification and functional characterization of the astacidin family of proline-rich host defence peptides (PcAst) from the red swamp crayfish (Procambarus clarkii, Girard 1852)

The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin

Thermal analysis of ice and glass transitions in insects that do and do not survive freezing

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline

Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35)

Autophagy: a critical regulator of cellular metabolism and homeostasis

A comparative genomics study on the effect of individual amino acids on ribosome stalling

Proline starvation induces unresolved ER stress and hinders mTORC1-Dependent tumorigenesis

Proline inhibits aggregation during protein refolding

Proline is a protein solubilizing solute

Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm

The Prolyl-tRNA synthetase inhibitor halofuginone inhibits SARS-CoV-2 infection

Identification of oral squamous cell carcinoma markers MUC2 and SPRR1B downstream of TANGO

Evaluation of proline analogs as trypanocidal agents through the inhibition of a proline transporter

Proline can be utilized as an energy substrate during flight of Aedes aegypti females

Familial hyperprolinemia, cerebral dysfunction and renal anomalies occurring in a family with hereditary nephropathy and deafness

The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction

Inactivation of the mouse L-Proline transporter PROT alters glutamatergic synapse biochemistry and perturbs behaviors required to respond to environmental changes

Renal tubular transport of proline, hydroxyproline, and glycine. 3. genetic basis for more than one mode of transport in human kidney

Structure of the mammalian antimicrobial peptide Bac7(1-16) bound within the exit tunnel of a bacterial ribosome

The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Genetic mapping to 10q23.3-q24.2, in a large Italian pedigree, of a new syndrome showing bilateral cataracts, gastroesophageal reflux, and spastic paraparesis with amyotrophy

Proline dehydrogenase: a key enzyme in controlling cellular homeostasis

Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes

Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes

TAp73 modifies metabolism and positively regulates growth of cancer stem-like cells in a redox-sensitive manner

In vitro alleviation of heavy metalinduced enzyme inhibition by proline

Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma

A new type antimicrobial peptide astacidin functions in antibacterial immune response in red swamp crayfish Procambarus clarkii

Structure and function of plant cell wall proteins

The fermentation analogy: a point of view for understanding the intriguing role of proline accumulation in stressed plants

Mitochondrial proline catabolism activates Ras1/cAMP/PKAinduced filamentation in Candida albicans

Further expansion of the phenotypic spectrum associated with mutations in ALDH18A1, encoding Delta(1)-pyrroline-5-carboxylate synthase (P5CS)

Cataracts, motor system disorder, short stature, learning difficulties, and skeletal abnormalities: a new syndrome?

New approaches and recent results concerning human-tissue collagen synthesis

Metabolic reprogramming during the Trypanosoma brucei life cycle

Osmotic shock induced protein destabilization in living cells and its reversal by glycine betaine

Small prolinerich proteins are cross-bridging proteins in the cornified cell envelopes of stratified squamous epithelia

Biochemical evidence that small proline-rich proteins and trichohyalin function in epithelia by modulation of the biomechanical properties of their cornified cell envelopes

Arasin 1, a proline-arginine-rich antimicrobial peptide isolated from the spider crab, Hyas araneus

Recovery from supercooling, freezing, and cryopreservation stress in larvae of the drosophilid fly

Genetic-and lifestyle-dependent dental caries defined by the acidic proline-rich protein genes PRH1 and PRH2

Spatially resolved metabolomics to discover tumor-associated metabolic alterations

Compatible solutes improve cryopreservation of human endothelial cells

Phytochemicals as regulators of genes involved in synucleinopathies

Proline: a multifunctional amino acid

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells

Renal iminoglycinuria without intestinal malabsorption of glycine and imino acids

Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system

The proline cycle as a potential cancer therapy target

Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans

The alphabet of intrinsic disorder: I. act like a Pro: on the abundance and roles of proline residues in intrinsically disordered proteins

Kinetics of osmotic stress regulate a cell fate switch of cell survival

Deciphering the mechanisms of intestinal imino (and amino) acid transport: the redemption of SLC36A1

The SLC36 family of protoncoupled amino acid transporters and their potential role in drug transport

Arginine can be synthesized from enteral proline in healthy adult humans

Arginine is synthesized from proline, not glutamate, in enterally fed human preterm neonates

The isolation of pure l-proline

Mitochondrial NADP(+) is essential for proline biosynthesis during cell growth

In vitro fertilisation of mouse oocytes in L-Proline and L-Pipecolic acid improves subsequent development

The plant oncogene rolD encodes a functional ornithine cyclodeaminase

From A. rhizogenes RolD to plant P5CS: exploiting proline to control plant development

Butyric acid induces spontaneous adipocytic differentiation of porcine bone marrow-derived mesenchymal stem cells

Synthesis of proline derived benzenesulfonamides: a potent anti-Trypanosoma brucei gambiense agent

Type II hyperprolinemia. Delta1-pyrroline-5-carboxylic acid dehydrogenase deficiency in cultured skin fibroblasts and circulating lymphocytes

Type 2 hyperprolinemia: absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity

O-glycosylated cell wall proteins are essential in root hair growth

Mammalian brain-specific L-proline transporter. neuronal localization of mRNA and enrichment of transporter protein in synaptic plasma membranes

Proline accumulation in plants: a review

Kidney amino acid transport

Proline metabolism and its implications for plant-environment interaction

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens

Effect of proline on lactate dehydrogenase activity: testing the generality and scope of the compatibility paradigm

L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture

Mechanisms mediating the biologic activity of synthetic proline, glycine, and hydroxyproline polypeptides in human neutrophils

A critical review: an overview of genetic influence on dental caries

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes

Alanine, proline and urea are major organic osmolytes in the snail Theodoxus fluviatilis under hyperosmotic stress

Identification of quenchers of photoexcited States as novel agents for skin photoprotection

Proline metabolism in the conceptus: implications for fetal growth and development

Polyamine synthesis from proline in the developing porcine placenta

Hydroxyprolinerich glycoproteins in plant reproductive tissues: structure, functions and regulation

Glutamyl-Prolyl-tRNA synthetase regulates proline-rich pro-fibrotic protein synthesis during cardiac fibrosis

Behavioral and neurochemical effects of proline

Autophagosome formation: core machinery and adaptations

Proline mediates metabolic communication between retinal pigment epithelial cells and the retina

Asymmetric michael addition of malonate anions to prochiral acceptors catalyzed by L-proline rubidium salt

Hydrogen peroxideinduced proline and metabolic pathway of its accumulation in maize seedlings

Pyrroline-5-carboxylate reductase 1 promotes cell proliferation via inhibiting apoptosis in human malignant melanoma

Incomplete proline catabolism drives premature sperm aging

Linkage and association analyses of schizophrenia with genetic variations on chromosome 22q11 in Koreans

Regulation of levels of proline as an osmolyte in plants under water stress

PYCR2 mutations cause a lethal syndrome of microcephaly and failure to thrive

Osmoprotection of Bacillus subtilis through import and proteolysis of proline-containing peptides

Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal

Proline concentration and its metabolism are regulated in a leaf age dependent manner but not by abscisic acid in pea plants exposed to cadmium stress

Knockdown of PYCR1 inhibits cell proliferation and colony formation via cell cycle arrest and apoptosis in prostate cancer

Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli

Cryobiological characteristics of L-proline in mammalian oocyte cryopreservation

Lproline: a highly effective cryoprotectant for mouse oocyte vitrification

Kindlin-2 acts as a key mediator of lung fibroblast activation and pulmonary fibrosis progression

Genome-Wide identification of PRP genes in apple genome and the role of MdPRP6 in response to heat stress

Mitochondrial NADP(H) generation is essential for proline biosynthesis

Novel inhibitors of the high-affinity L-proline transporter as potential therapeutic agents for the treatment of cognitive disorders

Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress

SlgA, encoded by the homolog of the human schizophrenia-associated gene PRODH, acts in clock neurons to regulate Drosophila aggression

We are most grateful to Prof. Maurizio Iaccarino for critical comments.

August 2021 | Volume 9 | Article 728576

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.