Proline Amino Acids: Properties, Function, Benefits, and Sources

What is proline?

Proline and its metabolite, hydroxyproline, are distinctive amino acids both chemically and biochemically. They represent one-third of amino acids in collagen proteins, which account for around 30% of body proteins. Proline has the highest demand for whole-body protein synthesis on a per-gram basis among all amino acids. The latest edition of Nutrient Requirements of Swine determined that l-proline (the physiological isomer) is unnecessary in the diets of gestating, neonatal, growing, finishing pigs, or boars to attain optimal production performance. Similarly, the National Research Council does not furnish data regarding proline concentrations in feed components typically utilized in the formulation of swine diets. Moreover, proline is not regarded as a nutritionally required or conditionally essential amino acid for humans in the absence of burns or injury. This is regrettable and indicates a deficiency in understanding proline biochemistry and nutrition in animals.

Metabolic disorders, proline metabolism, and nutrition have all attracted a lot of attention in the last several years. The importance of proline as a regulator of several cellular biochemical and physiological processes is becoming more and more demonstrated. For instance, in both humans and animals, proline serves as a signaling molecule, detects the energy state of cells, and contributes to redox reactions by generating pyrroline-5-carboxylate (P5C) and superoxide anion. Proline is also essential for the development and maturation of the conceptus (the fetus and its accompanying extraembryonic membranes) and for the differentiation of many cell types, including embryonic stem cells. The important function of proline in the nutrition of fetuses and newborns was reevaluated after the groundbreaking finding that it is a major amino acid (AA) in the production of polyamines in the placenta and small intestine. These polyamines regulate cell proliferation, differentiation, and DNA and protein synthesis. The field of animal and human nutrition is being influenced by new breakthroughs in proline metabolism. A survey of the most current findings in this promising field of AA research is the primary goal of this piece.

Proline structure

Proline possesses a secondary amine group, referred to as an imine, in lieu of a primary amine group. Consequently, proline is designated as an imino acid. The three-carbon R-group of proline is fused to the α-nitrogen group, resulting in a rotationally restricted rigid-ring structure. Consequently, prolyl residues in a polypeptide impose constraints on the folding of chains. In collagen, the primary protein of human connective tissue, specific prolyl residues undergo hydroxylation. Hydroxylation takes place during protein synthesis and necessitates ascorbic acid (vitamin C) as a cofactor. A deficiency of vitamin C results in the production of impaired collagen and leads to scurvy.

Because of its role as a tertiary amide in proteins and peptides, proline stands out among the conventional set of amino acids. That is why proline's amide bond isn't capable of donating hydrogen to support secondary structures like α-helices or β-sheets. Furthermore, the φ angle is severely limited by the cyclic side chain in proline, making it the sole classical amino acid with this property. Proteins often contain proline in certain locations, such as loops, turns, and flanking stable secondary-structure elements, which can be explained by its structural properties. Important structural or recognition motifs are frequently peculiar secondary-structure features of proline-rich peptides and proteins, such as polyproline PPI and PPII conformations. At long last, based on the propensity of the proline-formed amide bond to undergo cis/trans isomerization, the functional roles of the proline-N-flanking amide bond in cell event timing and signaling have been established. Considering the above, it's not surprising that a lot of work went into creating fluorine-containing labels that could replace the proline (Pro) residue in proteins and peptides and provide structural information at the locations where the Pro residue dictates function and structure.

The structure of proline. (Bhagavan N V., 2002)

Proline pKa

Proline possesses three principal pKa values indicative of the ionization of its functional groups: the carboxyl group (pKa ≈ 2.0), which dissociates a proton to yield a carboxylate anion; the amino group (pKa ≈ 9.0), which accepts a proton to generate a protonated amine; and the imino group (pKa ≈ 10.64) within its cyclic pyrrolidine ring, exhibiting an elevated pKa owing to the structural constraints of the ring. The pKa values alter proline's behavior in various pH conditions, influencing its involvement in protein folding and stability.

Molecular weight, pKa and logP. (Nie W., et al., 2018)

Proline charge

The charge of proline is contingent upon the pH of the surrounding environment. Under low pH (acidic circumstances), proline has a positive charge (+1) resulting from the protonation of both the carboxyl and amino groups. At neutral pH (about 7), it exists in a zwitterionic form, characterized by a deprotonated carboxyl group and a protonated amino group, yielding a net charge of zero (0). Under elevated pH (basic circumstances), proline possesses a negative charge (-1) due to the deprotonation of the amino groups while the carboxyl group stays deprotonated.

Proline amino acids at Creative Peptides

Proline synthesis and metabolism

Animals' ability to synthesize proline from amino acids arginine, ornithine, glutamine, and glutamate varies among species, tissues, and individual cells. The process of proline synthesis from arginine can be carried out by any mammalian tissue through the enzymes arginase (type I and type II), ornithine aminotransferase, and P5C reductase. The tissues that are quantitatively most active in this process are the kidneys, liver, small intestine (in postweaning animals), and mammary tissue. Proline, ornithine, and urea are the main byproducts of arginine catabolism in breast tissue. The lactating gland does not degrade arginine-derived proline since proline oxidase activity is not present in mammary tissue. This makes sure that the nursing mammary gland makes as much proline as possible from arginine. Breast tissue does not produce proline from glutamine or glutamate because it does not contain P5C synthase. The production of proline by nursing breast tissue is thus significantly impacted by arginase. Lactating mammary tissue has P5C reductase activity that is at least 50 times higher than P5C dehydrogenase activity, which prefers to convert arginine-derived P5C into proline instead of glutamate and glutamine. To stop the mammary tissue of breastfeeding pigs from losing all of its arginine carbons, the amino acid arginine is converted into proline. These results also provide light on the biochemical basis for the observation that the lactating mammary gland absorbs significantly more proline from plasma than arginine from sow's milk, but the reverse is true for arginine. Milk proteins are relatively rich in proline and relatively low in arginine due to the significant degradation of arginine for proline synthesis via the arginase pathway and the absence of proline catabolism in lactating swine mammary tissue.

The majority of tissues display proline oxidase activity, with the exception of breast tissue. Superoxide anion (O₂⁻), a result of this mitochondrial enzyme, has the potential to be transformed into hydrogen peroxide and other reactive oxygen species. Proline is the sole substrate for the synthesis of ornithine and, consequently, polyamines (putrescine, spermidine, and spermine) in cells and tissues (such as the swine placenta and neonatal pig enterocytes) that lack arginase activity. For two reasons: (1) the placenta and the neonatal small intestine develop at a tremendous rate, and (2) polyamines are important chemicals that control DNA and protein synthesis, cell proliferation, differentiation, and migration. Consequently, this has tremendous physiological and nutritional implications. To make up for the low levels of proline in the mother's blood, ruminants' placentas contain the enzymes arginase and proline oxidase.

Citrulline synthesis via P5C is very cell-and tissue-specific, despite the fact that all cells have the ability to recycle P5C into proline through P5C reductase and ornithine aminotransferase. The small intestine plays a distinct function in proline metabolism, and the fact that only enterocytes from mammals can synthesize citrulline from P5C is noteworthy. Extremely high arginase activity quickly hydrolyzes arginine into ornithine and urea, therefore the mammalian liver does not synthesize arginine netively, even though it can convert P5C into ornithine via the urea cycle. The enzyme P5C dehydrogenase can totally oxidize P5C to CO₂ in the kidneys and liver by forming α-ketoglutarate. The conversion of proline to carbon dioxide is extremely low in placentae and enterocytes that have low levels of P5C dehydrogenase. This optimizes the availability of P5C for the production of polyamines and prevents the irreversible loss of proline carbons. There is strong evidence that polyamines are crucial for the development, function, and health of the neonatal intestines.

The proline metabolic pathway. (Burke L., et al., 2020)

Proline functions

(1) Produce matrix collagens

Bone, cartilage, tendon, ligament, and interconnected fluid-filled CTs (which support and connect all other tissues, including epithelium and muscles) release collagens, which make up around 30% of all proteins in the human body. Having around 170 μM of L-Pro in plasma is crucial for collagen synthesis, and aberrant CT development can be caused by hereditary mutations in ALDH18A1 or PYCR1, which are involved in de novo L-Pro production. In order to maintain L-Pro homeostasis in humans, collagen deposition in rats, pigs, chickens, and fish, and bone density preservation in a mouse model of osteoporosis, extrinsic (dietary) L-Pro is crucial throughout adulthood.

(2) Produce antimicrobial peptides

The initial barrier against infections is the innate immune system, which produces L-Proline-rich antimicrobial peptides (PrAMPs)—a class of proteins released by insects, crustaceans, and mammals—that contain up to 50% L-Pro residues. The peptide antibiotic transporter SbmA is responsible for transporting PrAMPs into the bacterial cytoplasm, where they bind to ribosomal proteins and halt the creation of new proteins.

(3) Produce salivary proteins

About 70% of the proteins found in human saliva are unstructured L-Pro-rich salivary proteins (PRPs), which can contain as many as 40% L-Pro residues. Acidic (aPRP) and basic (bPRP) proteins are produced and secreted by the acinar cells of the parotid and submandibular salivary glands. The aPRPs bind calcium and shield the tooth surface, but the bPRPs bind polyphenols and tannins, creating an astringent feeling that affects food choices. It is tempting to assume that tannins in salivary glands can trigger a neutralizing response axis (ER stress→ATF4→L-Pro biosynthesis→PRP synthesis/secretion) because tannins cause ER stress and ATF4 expression, which in turn induces the transcription of L-Pro biosynthesis genes (ALDH18A1 and PYCR1).

(4) Energy provision

When glucose, fatty acids, or L-glutamine are oxidized, cellular energy in the form of ATP is produced. Nevertheless, there are cells that derive their energy from a Krebs cycle intermediate called α-KG, which is produced by oxidizing L-Pro in a three-stage process. Various cell types, including bacteria, insect muscle cells, and human cancer cells, can be sustained by up to 30 ATP equivalents per molecule of L-Pro. Note that there is no correlation between developmental delays and L-Pro oxidation genetic abnormalities in humans. This indicates that all normal human cells exclusively use L-Pro energy.

Proline structure, uses, biosynthesis and functions. (Patriarca E J., et al., 2021)

Proline application

(1) Amino acid supplement

Birds can only convert a little amount of arginine into proline because their tissues lack the arginase activity found in mammals. Thus, proline is an absolutely necessary amino acid for chickens and other birds. Furthermore, carnivores (such as ferrets and cats) do not have the enzyme P5C synthase in their cells, including enterocytes, hence they are unable to produce proline from glutamine and glutamate. Therefore, in these species, proline synthesis can only take place using arginine as a substrate. The absence of endogenous production of arginine and its high requirement for food sources make it a nutritionally necessary amino acid for carnivores. Due to the suppression of arginase by proline-derived ornithine, dietary supplementation with proline may compensate for some arginine in these animals.

(2) Provide osmoprotection

Shocks with a hypertonic potential cause water to drain out of cells, which decreases their volume and makes macromolecule stability worse. In response, cells store L-Pro, which causes water retention to occur in the reverse direction. Bacteria accumulate L-Pro by de novo L-Pro production, degradation of extracellular L-Pro-rich proteins, or the stimulation (up to 700-fold) of a low-affinity L-Pro transporter. Organisms like gastropod mollusks that live in environments that can change (fresh/brackish water, intertidal) rely on their capacity to store L-Pro. Concentrations of L-Pro can reach 60 mM (500 times greater than normal) in tomato cells as a response to dryness, salt, and cold. The buildup of L-Pro shields human cells against the harmful effects of hyperosmotic stress. The PP1 phosphatase subunit protein PPP1R15A/GADD34 promotes cis-to-trans Golgi trafficking and the plasma membrane localization of the SLC38A2 L-Pro transporter. L-Pro uptake aids in the recovery of viable cell volume after hypertonic stress.

(3) As a stress protectant

Yeast acquires resistance to ethanol and freezing when L-Pro accumulates. Approximately 80% of the entire pool of free amino acids in overwintering insects are L-Pro, which helps with hydration retention and cold resistance. It should be mentioned that the hyperprolinemic fly larvae of Chymomyza costata can withstand being submerged in liquid nitrogen (−196°C). The freezing tolerance and total body L-Pro concentration in fruit fly larvae are enhanced by feeding them a diet high in L-Pro.

The mechanism of azetidine-2-carboxylate (AZC) sensitivity or resistance. (Takagi H., 2008)

(4) Radical scavenging

L-Proline shields a variety of human cells from ROS-induced oxidative stress, including HEK293, HeLa, HepG2, Jurkat, BJAB, WM35, skin keratinocytes, and fibroblasts. It should be mentioned that the hydroxyl radicals (⋅OH) are stifled by the five-membered ring of the L-Pro molecule, which is called pyrrolidine or tetrahydropyrrole. L-Pro builds up in plants in reaction to oxidative chemicals and helps shield them from photo-oxidative stress, which is the production of reactive oxygen species (ROS) in response to light. The ability of proteins rich in L-Pro to neutralize reactive oxygen species (ROS), and more especially hydroxyl radicals.

(5) Heavy metal detoxification

Heavy metals like cadmium, chromium, and zinc accumulate in plants, and this helps immature olive plants and cultivated tobacco cells deal with the negative consequences of cadmium. The buildup of reactive oxygen species (ROS) is commonly linked to heavy metal poisoning. Apoptosis and aberrant ROS production are the results of L-Pro oxidation in mitochondria, which cadmium triggers via inducing p53, a transcriptional inducer of PRODH expression. Toxic metals like cadmium can start a cascade of events that includes p53, PRODH, ROS, and apoptosis.

(6) As the epigenetic modifier

The amounts of DNA 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) are both increased and decreased by supplementing with L-proline. This results in around 1,103 DNA double strand breaks (DMRs) spread out over all ESC chromosomes, with approximately 50% of these DMRs situated in gene promoter areas (mostly H) and about 20% in gene enhancers. Crucially, when exposed to VitC (50-150 μM) supplementation, almost 95% of genome locations that were hypermethylated after L-Pro supplementation become hypomethylated, suggesting that the same DNA regions are affected in opposing ways by the two epigenetic modifications. In order for TET demethylases to function, vitamin C is required. Additionally, cells that do not have TET-mediated DNA demethylase activity are likewise hypermethylated in around 90% of genomic areas that are hypermethylated when using a high L-Pro diet.

The addition of L-Proline to the diet causes a change in the H3K9 methylation status that affects over 1.6 × 104 sites in the genome, mostly in non-coding intergenic regions. Members of the JMJ dioxygenase family catalyze demethylation. When Jmjd1a (H3K9 demethylase) is silenced, ESCs undergo a molecular (upregulation of Fgf5 and Brachyury genes) and phenotypic (irregular flat-shaped colonies, sensitivity to trypsin digestion) transformation into pluripotent stem cells, comparable to what happens when a high L-Pro regimen is induced. How the abundance of L-Pro changes the methylation level of certain specific lysine residues (K9, K36) of histone H3 can be explained by differences in the expression level and/or kinetic parameters (substrate affinity) of distinct JMJs.

(7) Controls cell plasticity

A core of adherent cells surrounded by a crown of detachable cells displaying mesenchymal characteristics, such as long actin stress fibers and mature focal adhesion complexes, develops in flat-shaped cell colonies generated by embryonic stem cells sown at a low density (50-250 cells/cm²) in a high L-Pro regimen. In a completely reversible phenotypic shift called embryonic stem cell-to-mesenchymal transition (esMT), these L-Pro-induced cells are in a "metastable" equilibrium, dispersed from the colony center and quickly returning to re-establish adherent cell-cell connections. It is worth mentioning that E-cadherin is moved from the plasma membrane to the Golgi in detached cells. Interestingly, unlike in classical EMT, the CDH1 gene is not down-regulated during esMT.

Supplementing with L-Proline changes the expression of around 1.5 × 10³ protein-coding genes in naïve embryonic stem cells (ESCs), most of which are involved in cell adhesion, cell junction, and cell motility functions. This alters the transcriptome of the cells. In vitro, cells treated with L-Pro can develop into cardiomyocytes and neurons; they are also able to colonize mouse blastocysts (chimeric embryos), express pluripotency markers such as the Nanog homeobox, and are dependent on the leukemia inhibitory factor (LIF). The ability of L-Pro-treated ESCs to differentiate into primordial germ cell like cells (PGCLCs) and competent to form elongated gastruloids was recently reported by Cermola's team. This finding lends credence to the idea that an abundance of L-Pro pluripotentiates ESCs at an early stage.

Proline induces proliferation of stem and cancer cells. (Patriarca E J., et al., 2021)

FAQ

Is proline hydrophilic?

Because of its nonpolar side chain, proline is really thought of as hydrophobic, not hydrophilic. The pyrrolidine ring, which is five-membered and composed entirely of carbon and hydrogen atoms, is formed by proline's side chain. This structure is nonpolar because it does not contain any polar or charged groups.

Is proline polar or nonpolar?

The cyclic side chain of proline makes it very hydrophobic and nonpolar. Protein folding and structure are affected by its ability to introduce kinks and disrupt normal secondary structures such as α-helices and β-sheets.

Is proline aromatic?

Proline is not classified as aromatic. It possesses a five-membered cyclic side chain (pyrrolidine ring) consisting of carbon and nitrogen atoms. This structure is non-conjugated and lacks the properties of aromaticity. The side chain of proline is non-aromatic and has just saturated bonds, lacking the alternating double bonds typical of aromatic substances.

References

1. Wu G., et al., Proline and hydroxyproline metabolism: implications for animal and human nutrition, Amino acids, 2011, 40: 1053-1063.
2. Bhagavan N V. Medical biochemistry, Academic press, 2002.
3. Komarov I V., et al., Ulrich A S. 19F-Labeled amino acids for NMR structure analysis of membrane-bound peptides[M]//Fluorine in Life Sciences: Pharmaceuticals, Medicinal Diagnostics, and Agrochemicals. Academic Press, 2019: 349-395.
4. Nie W., et al., Small molecular weight aldose (d-Glucose) and basic amino acids (l-Lysine, l-Arginine) increase the occurrence of PAHs in grilled pork sausages, Molecules, 2018, 23(12): 3377.
5. Burke L., et al., The Janus-like role of proline metabolism in cancer, Cell death discovery, 2020, 6(1): 104.
6. Patriarca E J., et al., The multifaceted roles of proline in cell behavior, Frontiers in cell and developmental biology, 2021, 9: 728576.
7. Takagi H. Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications, Applied microbiology and biotechnology, 2008, 81(2): 211-223.