Proline is a non-essential amino acid, contributing to protein structure, osmoprotection, and redox balance. Its metabolism plays a pivotal role in collagen synthesis, energy homeostasis, and stress adaptation, linking to glutamate, arginine, and polyamine pathways. Dysregulation of proline metabolism is implicated in fibrosis, metabolic disorders, and cancer progression. This article explores proline's biochemical properties, metabolic pathways, and physiological functions in health and disease.
What is Proline?
Although proline is not classified as an essential amino acid, it plays a vital role in preserving protein structural integrity. Its molecular formula, C₅H₉NO₂, features a distinctive pyrrolidine ring that links to the amino group, creating a secondary amine-like configuration. This cyclic conformation restricts polypeptide chain flexibility, making proline indispensable for protein loops and turns. In collagen, its abundance enhances the stability of the triple-helix structure. Due to the absence of a conventional amide hydrogen, proline has limited hydrogen-bonding capacity and is predominantly found in regions of proteins with irregular architecture, where it disrupts α-helices.
Additionally, proline undergoes post-translational hydroxylation to form hydroxyproline (Hyp), a modification crucial for collagen integrity. This reaction requires vitamin C, and its deficiency leads to connective tissue disorders such as scurvy. Overall, proline's distinctive structural properties underpin its essential roles in protein folding, osmoregulation, stress responses, and metabolic regulation.
Figure 1. Proline structure.
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Types and Modifications of Proline
During protein synthesis, proline functions through its unique backbone structure, rather than relying on the characteristics of its side chain. Unlike other α-amino acids, the five-membered ring of proline restricts the conformation of its N-atom, leading to the coexistence of cis and trans isomers. This property is crucial for the secondary structure of proteins, particularly the stability of β-turns and the collagen triple helix.
(1) Natural Proline
Proline primarily exists in two forms: L-proline (L-Pro) and D-proline (D-Pro). L-proline is the common form found in natural proteins, participating in protein synthesis and playing important roles in amino acid metabolism, wound healing, and oxidative stress responses. D-proline is relatively rare and is primarily found in certain bacteria, fungi, and their metabolites, such as bacterial cell wall peptidoglycans and non-ribosomal peptides.
(2) Major Modifications
Proline undergoes several post-translational modifications that affect protein function and stability, including:
- Hydroxyproline (Hyp): There are two main forms—4-hydroxyproline (4-Hyp) and 3-hydroxyproline (3-Hyp). 4-Hyp enhances the stability of collagen's triple helix and is widely distributed in connective tissues, cartilage, and extracellular matrix proteins. 3-Hyp is primarily found in type IV collagen, stabilizing the basement membrane structure.
- 5-Hydroxyproline: This form is rarer and is mainly found in certain microbial metabolites and natural antibiotics, potentially affecting molecular interactions and antimicrobial activity.
- Phosphorylated Proline: Though relatively rare, phosphorylated proline has been found in certain signaling pathways and regulatory proteins. Phosphorylation of proline may influence protein conformation, interactions, and function, thereby regulating cellular signaling, metabolism, and stress responses.
Physiological Functions of Proline
Proline has a variety of physiological functions within organisms, primarily reflected in the following aspects:
(1) Structural Stability
Proline is important for collagen, and its cyclic structure restricts the rotational freedom of the peptide chain, enhancing the stability of the triple helix and increasing mechanical strength and pressure resistance. Collagen is widely present in connective tissues such as skin, cartilage, bones, tendons, and blood vessels, and proline is critical for maintaining the structural integrity of these tissues.
(2) Immune Regulation and Inflammatory Response
Proline metabolites can influence T cell differentiation and regulate inflammatory responses. For example, proline and its derivatives can act as signaling molecules, modulating cytokine levels and thus affecting immune responses. Additionally, proline plays a role in antioxidant defense mechanisms, increasing cell survival under oxidative stress.
(3) Energy Metabolism and Stress Response
Proline is oxidized by proline dehydrogenase (PRODH) to produce Δ¹-pyrroline-5-carboxylate (P5C), which is further metabolized into glutamate and enters the tricarboxylic acid (TCA) cycle to provide energy. Therefore, proline not only serves as a vital component of protein synthesis but also as an energy source, supporting cell growth under conditions of nutrient deprivation or oxidative stress. Furthermore, proline plays an important role in osmoregulation and cellular adaptive regulation, especially in plant stress resistance and certain microbial survival strategies.
Figure 2. Proline multi-function. (A. Bahadur, et al., 2011)
Proline Metabolism Process
Proline metabolism mainly occurs through oxidative pathways, involving oxidation, reduction, and phosphorylation processes. In the mitochondria of various tissues, proline is oxidized by proline dehydrogenase (PRODH) into pyrroline-5-carboxylate (P5C), transferring electrons to the electron transport chain to promote ATP production. P5C is then catalytically converted into glutamate by pyrroline-5-carboxylate dehydrogenase (P5CDH), and glutamate enters the TCA cycle to provide energy or to be used for amino acid synthesis. Proline metabolism is closely related to cellular redox homeostasis and stress responses; accumulated proline acts as an osmoprotectant and antioxidant, aiding in the maintenance of cellular homeostasis. Ultimately, excess proline is metabolized in the liver and excreted from the body through nitrogenous waste.
Proline-Pyrroline-5-Carboxylate (P5C) Cycle
The proline-pyrroline-5-carboxylate (P5C) cycle is crucial in proline metabolism, not only regulating energy metabolism but also involving redox homeostasis, stress responses, and signal transduction. In this cycle, proline is oxidized to P5C, which promotes ATP synthesis through the mitochondrial electron transport chain. Once P5C is converted into glutamate, it can further enter the TCA cycle for energy production or serve as a precursor for amino acids and other molecules. Under oxidative stress, P5C is reduced back to proline, enhancing antioxidant capacity. The reversibility of this cycle helps maintain the NADH/NAD+ and NADPH/NADP+ balance, regulating cellular redox status. Additionally, proline dehydrogenase (PRODH) is regulated by p53, affecting apoptosis, proliferation, and differentiation processes. The P5C cycle holds significant research value in cancer, neurodegenerative diseases, and stress-adaptive responses.
Figure 3. The metabolic pathways of proline.
Proline Metabolism and Health
Proline metabolism regulates energy homeostasis, redox balance, and cellular stress responses through the P5C cycle, affecting metabolic syndrome, cardiovascular health, and nervous system function. Studies suggest that proline metabolism influences mitochondrial function, oxidative balance, and potentially affects insulin sensitivity, lipid metabolism, and inflammatory responses, thereby impacting metabolic syndrome. Proline metabolism helps reduce reactive oxygen species (ROS) accumulation, lower oxidative stress and inflammation, and improve vascular function, which may reduce the risk of cardiovascular diseases. Additionally, proline plays a role in maintaining nervous system health by enhancing antioxidant capacity, reducing oxidative damage, and regulating glutamate and γ-aminobutyric acid (GABA) levels, affecting neural plasticity and cognitive function. However, its precise mechanisms require further investigation.
Proline's Potential Anti-inflammatory Effect
The potential anti-inflammatory and antioxidant roles of proline have been demonstrated. One study indicated that proline could counteract lipopolysaccharide (LPS)-induced inflammation and oxidative stress in the cerebral cortex and cerebellum of rats. The experiment found that combined proline treatment effectively prevented the upregulation of inflammatory markers such as S100B and GFAP, reduced oxidative stress, and restored energy metabolism enzyme functions, showing significant anti-inflammatory and antioxidant effects.
Proline Metabolism and Tumor Development
Proline metabolism plays a complex dual role in tumor progression and inflammation regulation, where changes in the activity of key enzymes (such as P5CS, PYCR, and PRODH) have profound effects on the tumor microenvironment and immune responses. In a glutamine-limited tumor environment, cancer cells preferentially utilize glutamate to synthesize glutamine by downregulating the rate-limiting enzyme P5CS, maintaining nucleotide and asparagine synthesis to support tumor proliferation (Nature Metabolism). This metabolic plasticity enables cancer cells to survive under nutritional stress, and inhibiting P5CS could enhance sensitivity to GS inhibitors, suggesting that targeting proline metabolism may improve the efficacy of existing therapeutic strategies.
Proline Metabolism and the Tumor Microenvironment
Proline metabolism regulates tumor progression via cancer-associated fibroblasts (CAFs). CAFs highly express PYCR1, which promotes proline synthesis and supports collagen deposition, forming a dense extracellular matrix (ECM) that hinders the penetration of anti-tumor drugs and promotes tumor metastasis (Current Opinion in Biotechnology). Additionally, proline metabolism plays a crucial role in inflammation by modulating reactive oxygen species (ROS) levels. ROS generated by PRODH catalyzing proline degradation activate the NF-κB pathway, promoting the secretion of pro-inflammatory cytokines such as IL-6 and IL-4, which induce macrophage polarization towards the M2 phenotype. Meanwhile, proline metabolism intermediates, P5C and ornithine, suppress T cell activity, creating an immunosuppressive microenvironment that weakens anti-tumor immune responses.
Tumor Heterogeneity and Proline Metabolism
The role of proline metabolism in different cancers shows significant heterogeneity. In pancreatic cancer, PRODH supports the TCA cycle by breaking down collagen-derived proline, promoting tumor growth. In lung cancer, high expression of PRODH activates pro-inflammatory signaling through ROS production, driving tumor progression. In contrast, PYCR1 is highly expressed in various cancers, such as breast cancer and renal cancer, and is associated with poor prognosis. It alleviates oxidative stress by maintaining the NAD(P)H/NAD(P)+ balance and promotes protein synthesis via the Akt/mTOR pathway. The differences in metabolic pathways suggest that targeted therapies should be tailored based on the specific metabolic characteristics of each tumor type. In summary, proline metabolism regulates energy metabolism, ECM remodeling, and the immune microenvironment, becoming a key node in tumor progression and inflammation. This provides new perspectives for understanding tumor metabolic mechanisms.
Conclusion
As a structurally unique amino acid, proline plays indispensable roles in biological systems through its distinctive molecular properties and versatile metabolic functions. The cyclic structure of proline not only stabilizes collagen and other extracellular matrix proteins but also modulates protein folding dynamics, directly influencing tissue integrity and mechanical resilience. Beyond its structural contributions, proline participates in critical physiological processes, including energy metabolism, oxidative stress response, and immune regulation, with its metabolic intermediates serving as key regulators of redox homeostasis and cellular adaptation.
The dual roles of proline metabolism in health and disease highlight its biological complexity. While supporting wound healing, neurological function, and antioxidant defense, proline metabolism also exhibits context-dependent effects in pathological conditions such as cancer. Tumor cells exploit proline metabolic plasticity to fuel proliferation under nutrient stress, while cancer-associated fibroblasts leverage proline synthesis to remodel the tumor microenvironment. This metabolic duality extends to inflammatory regulation, where proline-derived metabolites may either mitigate oxidative damage or paradoxically promote immunosuppressive signaling.
Ongoing research continues to unravel the intricate networks connecting proline metabolism with systemic physiology and disease mechanisms. The tissue-specific expression of metabolic enzymes like PRODH and PYCR1, coupled with their divergent roles across cancer types, underscores the need for precision therapeutic strategies. Future investigations into the crosstalk between proline metabolism, extracellular matrix dynamics, and immune modulation could yield novel biomarkers and targeted interventions. Such advancements promise to translate fundamental insights into clinical applications, potentially revolutionizing approaches to tissue repair, metabolic disorders, and cancer therapy. This evolving understanding positions proline not merely as a structural amino acid but as a dynamic metabolic nexus with far-reaching implications for human health.
References
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- Kay EJ, Zanivan S, Rufini A: Proline metabolism shapes the tumor microenvironment: from collagen deposition to immune evasion. Current Opinion in Biotechnology 2023, 84:103011. https://doi.org/10.1016/j.copbio.2023.103011
- Linder SJ, Bernasocchi T, Martínez-Pastor B, Sullivan KD, Galbraith MD, Lewis CA, Ferrer CM, Boon R, Silveira GG, Cho HM et al: Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells. Nature Metabolism 2023, 5(12):2131-2147.10.1038/s42255-023-00919-3. https://doi.org/10.1038/s42255-023-00919-3
