Perspectives, past, present and future: the proline cycle/proline-collagen regulatory axis.

In the 35 years since the introduction of the "proline cycle", its relevance to human tumors has been widely established. These connections are based on a variety of mechanisms discovered by many laboratories and have stimulated the search for small molecule inhibitors to treat cancer or metastases. In addition, the multi-layered connections of the proline cycle and the role of proline and hydroxyproline in collagen provide an important regulatory link between the extracellular matrix and metabolism.

Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate.

Proline dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I-IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration.

Proline metabolism and cancer: emerging links to glutamine and collagen.

PURPOSE OF REVIEW: Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism. RECENT FINDINGS: The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production, storage and mobilization. SUMMARY: Mechanisms by which the proline regulatory axis modulates the cancer phenotype are being revealed. Proline can be synthesized from glutamine as well as derived from collagen degradation. The metabolism of proline serves as a source of energy during stress, provides signaling reactive oxygen species for epigenetic reprogramming and regulates redox homeostasis.

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

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ(1)-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using (13)C,(15)N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies.

The regulatory mechanisms of proline and hydroxyproline metabolism: Recent advances in perspective.

For diverse human tumors, growth and metastasis are dependent on proline synthesis, but the mechanisms underlying this association are not clear. Proline incorporated into collagen is primarily synthesized from glutamine. Thus, rates of collagen synthesis are modulated by the enzymes of proline synthesis. On the other hand, the hydroxylation of collagen proline requires αKG, ascorbate and ferrous iron, substrates necessary for the epigenetic demethylation of DNA and histones. The metabolic relationship of proline and hydroxyproline degradation are initiated by distinct dehydrogenases but the respective oxidized products, P5C and OH-P5C are substrates for P5C Reductase and P5C Dehydrogenase allowing for mutual competition. This provides a model by which proline synthesis in cancer plays a role in reprogramming gene expression. The metabolism of proline and hydroxyproline are also linked to the HIF response to hypoxia. Hypoxia increased the expression of ALDH18A1, which is the limiting step in proline and collagen synthesis. Hydroxyproline increases levels of HIF-1α presumably by inhibiting its degradation. These new findings allow the suggestion that there is a regulatory axis from glutamine to proline and collagen synthesis, and the release of free hydroxyproline can feed back on the HIF pathway.

Proline metabolism and microenvironmental stress.

Proline, the only proteinogenic secondary amino acid, is metabolized by its own family of enzymes responding to metabolic stress and participating in metabolic signaling. Collagen in extracellular matrix, connective tissue, and bone is an abundant reservoir for proline. Matrix metalloproteinases degrading collagen are activated during stress to make proline available, and proline oxidase, the first enzyme in proline degradation, is induced by p53, peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands, and by AMP-activated protein kinase downregulating mTOR. Metabolism of proline generates electrons to produce ROS and initiates a variety of downstream effects, including blockade of the cell cycle, autophagy, and apoptosis. The electrons can also enter the electron transport chain to produce adenosine triphosphate for survival under nutrient stress. Pyrroline-5-carboxylate, the product of proline oxidation, is recycled back to proline with redox transfers or is sequentially converted to glutamate and alpha-ketoglutarate. The latter augments the prolyl hydroxylation of hypoxia-inducible factor-1alpha and its proteasomal degradation. These effects of proline oxidase, as well as its decreased levels in tumors, support its role as a tumor suppressor. The mechanism for its decrease is mediated by a specific microRNA. The metabolic signaling by proline oxidase between oxidized low-density lipoproteins and autophagy provides a functional link between obesity and increased cancer risk.

Developmental cardiac hypertrophy in a mouse model of prolidase deficiency.

BACKGROUND: Hypertrophic cardiomyopathy, characterized by thickened ventricular walls and reduced ventricular chamber volume, is a common cause of sudden cardiac death in young people. Most inherited forms result from mutations in genes encoding sarcomeric proteins. METHODS: Histologic analysis identified embryonic cardiac hypertrophy in dark-like mutant mice. BrdU analysis was performed to measure proliferation and cardiomyocytes were isolated to measure cell size. The dark-like mutation was identified by positional cloning. RESULTS: The dark-like mutation causes cardiomyocyte hypertrophy due to loss-of-function of peptidase d (Pepd), which encodes prolidase, a cytosolic enzyme that recycles proline for collagen re-synthesis. Prolidase deficiency is a rare autosomal recessive disease in humans with a broad phenotypic spectrum not reported to include heart defects, but a conserved role for prolidase in heart development was confirmed by morpholino knockdown in zebrafish. We tested the hypothesis that loss of prolidase function disrupts collagen-mediated integrin signaling and determined that the levels of several key integrin transducers were reduced in the hearts of dark-like mutant embryos. CONCLUSIONS: This work identifies dark-like mice as a model of prolidase deficiency that will be valuable for studying the role of proline metabolism in normal physiology and disease processes, and suggests that integrin signaling may regulate the onset of hypertrophic cardiac growth.

Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

Proline Metabolism in Tumor Growth and Metastatic Progression.

Cancer cells show a formidable capacity to survive under stringent conditions, to elude mechanisms of control, such as apoptosis, and to resist therapy. Cancer cells reprogram their metabolism to support uncontrolled proliferation and metastatic progression. Phenotypic and functional heterogeneity are hallmarks of cancer cells, which endow them with aggressiveness, metastatic capacity, and resistance to therapy. This heterogeneity is regulated by a variety of intrinsic and extrinsic stimuli including those from the tumor microenvironment. Increasing evidence points to a key role for the metabolism of non-essential amino acids in this complex scenario. Here we discuss the impact of proline metabolism in cancer development and progression, with particular emphasis on the enzymes involved in proline synthesis and catabolism, which are linked to pathways of energy, redox, and anaplerosis. In particular, we emphasize how proline availability influences collagen synthesis and maturation and the acquisition of cancer cell plasticity and heterogeneity. Specifically, we propose a model whereby proline availability generates a cycle based on collagen synthesis and degradation, which, in turn, influences the epigenetic landscape and tumor heterogeneity. Therapeutic strategies targeting this metabolic-epigenetic axis hold great promise for the treatment of metastatic cancers.

Regulation and function of proline oxidase under nutrient stress.

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and alpha-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels.

Proline Metabolism in Cell Regulation and Cancer Biology: Recent Advances and Hypotheses.

SIGNIFICANCE: It is increasingly clear that proline metabolism plays an important role in metabolic reprogramming, not only in cancer but also in related fields such as aging, senescence, and development. Although first focused on proline catabolism, recent studies from a number of laboratories have emphasized the regulatory effects of proline synthesis and proline cycling. Recent Advances: Although proline dehydrogenase/proline oxidase (PRODH/POX) has been known as a tumor protein 53 (P53)-activated source of redox signaling for initiating apoptosis and autophagy, senescence has been added to the responses. On the biosynthetic side, two well-recognized oncogenes, c-MYC and phosphoinositide 3-kinase (PI3K), markedly upregulate enzymes of proline synthesis; mechanisms affected include augmented redox cycling and maintenance of pyridine nucleotides. The reprogramming has been shown to shift in clonogenesis and/or metastasis. CRITICAL ISSUES: Although PRODH/POX generates reactive oxygen species (ROS) for signaling, the cellular endpoint is variable and dependent on metabolic context; the switches for these responses remain unknown. On the synthetic side, the enzymes require more complete characterization in various cancers, and demonstration of coupling of proline metabolites to other pathways may require studies of protein-protein interactions, membrane transporters, and shuttles. FUTURE DIRECTIONS: The proline metabolic axis can serve as a scaffold on which a variety of regulatory mechanisms are integrated. Once understood as a central mechanism in cancer metabolism, proline metabolism may be a good target for adjunctive cancer therapy.

Extracellular matrix and HIF-1 signaling: the role of prolidase.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in stress-responsive gene expression. Although primarily sensitive to hypoxia, HIF-1 signaling can be regulated by a number of stress factors including metabolic stress, growth factors and molecules present in the extracellular matrix (ECM). Degradation of ECM by metalloproteinases (MMP) is important for tumor progression, invasion and metastasis. ECM is predominantly collagen, and the imino acids (Pro and HyPro) comprise 25% of collagen residues. The final step in collagen degradation is catalyzed by prolidase, the obligate peptidase for imidodipeptides with Pro and HyPro in the carboxyl terminus. Defective wound healing in patients with inherited prolidase deficiency is associated with histologic features of angiopathy suggesting that prolidase may play a role in angiogenesis. Because HIF-1 alpha is central to angiogenesis, we considered that prolidase may modulate this pathway. To test this hypothesis, we made expression constructs of human prolidase and obtained stable transfectants in colorectal cancer cells (RKO). Overexpression of prolidase resulted in increased nuclear hypoxia inducible factor (HIF-1 alpha) levels and elevated expression of HIF-1-dependent gene products, vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). The activation of HIF-1-dependent transcription was shown by prolidase-dependent activation of hypoxia response element (HRE)-luciferase expression. We used an oxygen-dependent degradation domain (ODD)-luciferase reporter construct as a surrogate for HIF-1 alpha as an in situ prolyl-hydroxylase assay. Since this reporter is degraded by VHL-dependent mechanisms, the increased levels of luciferase observed with prolidase expression reflected the decreased HIF-1 alpha prolyl hydroxylase activity. Additionally, the differential expression of prolidase in 2 breast cancer cell lines showed prolidase-dependent differences in HIF-1 alpha levels. These findings show that metabolism of imidodipeptides by prolidase plays a previously unrecognized role in angiogenic signaling.

The metabolism of proline as microenvironmental stress substrate.

Proline, a unique proteogenic secondary amino acid, has its own metabolic system with special features. Recent findings defining the regulation of this system led us to propose that proline is a stress substrate in the microenvironment of inflammation and tumorigenesis. The criteria for proline as a stress substrate are: 1) the enzymes utilizing proline respond to stress signaling; 2) there is a large, mobilizable pool of proline; and 3) the metabolism of proline serves special stress functions. Studies show that the proline-utilizing enzyme, proline oxidase (POX)/proline dehydrogenase (PRODH), responds to genotoxic, inflammatory, and nutrient stress. Proline as substrate is stored as collagen in extracellular matrix, connective tissue, and bone and it is rapidly released from this reservoir by the sequential action of matrix metalloproteinases, peptidases, and prolidase. Special functions include the use of proline by POX/PRODH to generate superoxide radicals that initiate apoptosis by intrinsic and extrinsic pathways. Under conditions of nutrient stress, proline is an energy source. It provides carbons for the tricarboxylic acid cycle and also participates in the proline cycle. The latter, catalyzed by mitochondrial POX and cytosolic pyrroline-5-carboxylate reductase, shuttles reducing potential from the pentose phosphate pathway into mitochondria to generate ATP and oxidizing potential to activate the cytosolic pentose phosphate pathway.

The metabolism of proline, a stress substrate, modulates carcinogenic pathways.

The resurgence of interest in tumor metabolism has led investigators to emphasize the metabolism of proline as a "stress substrate" and to suggest this pathway as a potential anti-tumor target. Proline oxidase, a.k.a. proline dehydrogenase (POX/PRODH), catalyzes the first step in proline degradation and uses proline to generate ATP for survival or reactive oxygen species for programmed cell death. POX/PRODH is induced by p53 under genotoxic stress and initiates apoptosis by both mitochondrial and death receptor pathways. Furthermore, POX/PRODH is induced by PPARgamma and its pharmacologic ligands, the thiazolidinediones. The anti-tumor effects of PPARgamma may be critically dependent on POX/PRODH. In addition, it is upregulated by nutrient stress through the mTOR pathway to maintain ATP levels. We propose that proline is made available as a stress substrate by the degradation of collagen in the microenvironmental extracellular matrix by matrix metalloproteinases. In a manner analogous to autophagy, this proline-dependent process for bioenergetics from collagen in extracellular matrix can be designated "ecophagy".

Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides.

The metabolism of the nonessential amino acid proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ(1)-pyrroline-5-carboxylate (P5C) or proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of proline was blocked by concomitant proline dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between proline and P5C rather than product proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy.

Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development.

PURPOSE: In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. METHODS: We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. RESULTS: We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. CONCLUSIONS: Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.

Lack of prolidase causes a bone phenotype both in human and in mouse.

The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and µCT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest.