Pyrroline 5 Carboxylate Reductase - an overview (2023)

1.02.3.1.2 Proline, ornithine, and arginine are synthesized from glutamate

Proline and arginine are both derived from glutamate. γ-Glutamyl kinase catalyzes the first step in this process, which involves the activation of the γ-carboxylate group of glutamate by phosphorylation with adenosine triphosphate (ATP). This forms the γ-glutamyl phosphate intermediate that is reduced to glutamate-5-semialdehyde. Glutamate-5-semialdehyde spontaneously cyclicizes to an internal Schiff base. The final reduction to proline is catalyzed by pyrroline-5-carboxylate reductase, which requires the presence of either reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

The formation of the semialdehyde is a branch point with one branch leading to proline, as previously described and the other leading to the formation of ornithine and arginine. In humans, glutamate-5-semialdehyde is directly transaminated to yield ornithine in a reaction catalyzed by ornithine-δ-aminotransferase. Ornithine is then converted into arginine through the urea cycle. The pathway for Escherichia coli likewise involves ATP-dependent reduction of the carboxyl group of glutamate to an aldehyde, which is then converted to its corresponding amine by transamination. Hydrolysis of the acetyl protecting group eventually forms ornithine, which, as previously mentioned, is converted into arginine via the urea cycle.

6 Notes and considerations

Note 1: Design a plasmid and cloning scheme that enables expression of both the POI and the E. coli ProRS. We typically use inducible expression of the POI (e.g., via the IPTG-inducible T5 promoter), and ProRS over-expression with its endogenous promoter. Both proteins can be expressed from the same plasmid backbone; see Fig. 3 for the scheme of the final construct described here. We have also found success with different construct designs (alternative vector backbones, promoters, two plasmid approaches, etc.) that facilitate expression of both the POI and the ProRS. Cloning approaches other than those described here may be more convenient, depending upon the situation.

Note that ProRS over-expression may not be necessary for the incorporation of some ncPro analogs with particularly high incorporation efficiencies; for example, 3R-F, 3S-F, 4R-F, 4S-F, and Dhp are reported to have been incorporated without ProRS over-expression (Kim et al., 2004, 2006). However, as there are conflicting reports in different expression systems (for example, Lukesch et al., 2019), we recommend ProRS over-expression in general.

Note 2: Obtain an appropriate strain of E. coli for expression of the POI. A bacterial strain unable to synthesize proline (i.e., a proline auxotroph) is necessary for efficient proline analog incorporation into the recombinant POI. Strains deficient in proline biosynthesis via disruption of proA, proB or proC are available from the Coli Genetic Stock Center (CGSC, https://cgsc.biology.yale.edu). We generally use the strain CAG18515; however, we have also had success with other strains (such as those from the Keio collection).

In some cases, authors have noted oxidative degradation of the proline analogs Thz and Dhp via l-proline dehydrogenase (putA) and Δ1-pyrroline-5-carboxylate reductase (proC). Strain UMM5, which contains mutations in both of these genes, resulted in enhanced incorporation of these ncPro residues (Kim et al., 2004).

Note 3: Optimize expression conditions for the POI in M9 medium using the proline auxotroph of choice. These efforts typically involve screening parameters such as expression time, temperature, and inducer concentrations. Some reports in the literature have suggested that osmolyte identity (e.g., NaCl vs sucrose) and concentration can influence ncPro incorporation efficiencies and protein yield (for example, Buechter et al., 2003; Kim et al., 2004), and so we also suggest screening these parameters in the relevant expression system. Relative protein expression levels can be evaluated by methods such as SDS-PAGE, and extent of ncPro replacement by MALDI-TOF.

Note 4: Determine a method of purification for the POI. One approach often used to isolate a hexahistidine-tagged protein (common for proteins encoded on a pQE-80L vector) is IMAC using Ni-NTA agarose resin (e.g., HisPur Ni-NTA Resin, Thermo Fisher Scientific). In this case, E. coli cells are lysed with B-PER Complete after expression. Hexahistidine-tagged proinsulin was isolated from the inclusion body fraction by IMAC purification performed under denaturing conditions (8M urea), using a low pH (3.0) elution buffer. However, purification methods will likely vary depending upon the POI and affinity tag used. For example, denaturants such as urea are typically not used if the POI is soluble; instead, the protein is purified under native conditions. Resources that describe protein purification protocols are available; for instance, we have found the QIAexpressionist (which can be found on qiagen.com) to be helpful in planning purification of hexahistidine-tagged proteins.

Note 5: Digestion of the POI and MALDI-TOF analysis of the resulting peptide fragments are often necessary to permit accurate determination of ncPro incorporation efficiencies. The protocol here describes digestion with the peptidase Glu-C; however, the choice of peptidase will depend on the sequence of the POI. We recommend using a peptidase that results in at least one peptide fragment that contains only one proline residue. This makes the analysis of proline analog incorporation more straightforward.

15.4.2.4 Abiotic Stress Response: The Metabolomes

In salt-stressed M. crystallinum, a succulent desert halophyte and CAM plant, extensive alteration in metabolome was observed in metabolic pathways of compatible solutes, sugars, sugar alcohols, protein and nonprotein amino acids, and organic acids (Barkla and Vera-Estrella, 2015). One nonprotein amino acid, pipecolic acid, known to be a critical regulator of inducible plant immunity and resistance to bacterial pathogens, changed 12-fold in salt-stressed plants. Out of 194 known metabolites in the epidermal bladder cell, significant changes were observed in 57 proteins (30%) upon salt treatment. This and previous transcriptomic studies on epidermal bladder cells have revealed an increase in transcripts encoding myo-inositol-1-phosphate synthase and myo-inositol O-methyltransferase 1, the two key enzymes in the pathway leading to pinitol synthesis via ononitol, and delta-1-pyrroline-5-carboxylate synthase and pyrroline-5-carboxylate reductase, the two proline biosynthetic enzymes were highly abundant due to their significant upregulation in the epidermal bladder cell transcriptome (up to 170-fold) under salinity (Barkla and Vera-Estrella, 2015; Oh et al., 2015). Metabolites such as organic acids (malate, citrate, fumarate), amino acids (valine asparagines), sinapyl alcohol, and components involved in ROS metabolism such as ascorbate, glutathione, and antioxidant enzymes as well as fatty alcohol dodeconol, fatty acid hydrolase, involved in the biosynthesis of cuticular wax have been changed, as similarly observed in Arabidopsis and tobacco trichomes, legume Lotus creticus, and the salt-tolerant Arabidopsis relative Thellungiella salsuginea (Barkla and Vera-Estrella, 2015; Oh et al., 2015). Metabolomes in Arabidopsis and the legume Vicia faba guard cell and Bradyrhizobium japonicum infected soybean root hair in separate investigations include ROS, nitric oxide, flavonoids, trehalose, carboxylic acids, abscisic acid, and auxin as important components of the signaling cascade controlling the stomatal movements in response to osmotic stresses, pathogenic organisms Pseudomonas syringae, and establishment of the symbiosis process, respectively (Brechenmacher et al., 2010; Ou et al., 2014; Misra et al., 2015). A lipidomic study in Commelina communis and A. thaliana guard cells also detected fatty acids as the key regulator of stomatal response to both biotic and abiotic stresses (Ou et al., 2014; Misra et al., 2015).

3.5 Proline

Proline is a key proteinogenic amino acid that is highly abundant in the extracellular matrix component collagen [170]. Proline is synthesized via pyrroline-5-carboxylate reductases and degraded by a mitochondrial enzyme, proline dehydrogenase (PRODH). Both de novo synthesis and degradation of proline involve metabolite intermediate D1-pyrroline-5-carboxylic acid (P5C) and are coupled to cellular energy and redox status, demonstrating the additional functions of this amino acid. During proline degradation, PRODH bound to the mitochondrial inner membrane transfers electrons from proline to a FAD⁺ prior to its transfer to a coenzyme Q pool, enabling ATP production through ETC [171]. In the de novo synthesis of proline, the final step that converts P5C into proline catalyzed by mitochondrial PYCRs (PYCR1 and PYCR2) or cytosolic PYCRL requires reducing the power of NADPH or NADH, depending on the specific isoform [172].

Several cancers depend on the uptake of exogenous proline due to a partial auxotrophy. For example, a human leukemic lymphoblastoid cell line, REH, presents a defect in PYCR activity and completely depends on taking up proline to proliferate [173]. A recent study identified a group of pancreas and lung cancer cell lines as dependent on extracellular proline supplementation [174]. These proline-dependent cancer cell lines exhibited a basal metabolic shunting of glutamine into proline synthesis but failed to trigger the de novo synthesis pathway upon proline depletion. This dependency on exogenous proline was recapitulated invivo using a proline-free diet [174]. Notably, c-Myc is a known regulator of proline metabolism and plays an important role in activating proline metabolism genes in Burkitt's lymphoma and prostate cancer models [175]. Despite these results, the determinants of proline dependency invivo have not yet been identified.

Similar to aspartate, proline levels may also limit tumor formation. Ribosome profiling studies to identify tRNA abundance in human clear cell renal cell carcinoma (ccRC) samples revealed enriched signals in proline codons, indicating a proline limitation invivo [20]. Interestingly, the limitation of proline correlated with an increase in the expression of the de novo proline synthesis enzyme PYCR1, and its knockdown in different ccRC cell lines strongly impaired invivo tumor formation [20]. These results suggest that proline can limit some primary tumors. Interestingly, matrix metalloproteinases (MMP) can degrade collagen [176], the most abundant protein in the body, and increase proline availability. Because MMP-mediated degradation of ECM is considered pro-tumorigenic in many contexts [177], this raises the possibility that cancer-associated MMPs may provide an important source of proline and thus make tumors less dependent on their synthesis. The energy and redox status can also determine the metabolic route by which cancer cells obtain their proline [178]. For example, an imbalance of the cellular redox state triggered by IDH1 mutation can impact proline metabolism. Indeed, IDH1-mutant glioma cells compensate for this imbalance by increasing proline biosynthesis, in which enhanced PYCR1 activity maintains redox homeostasis [179].

A higher demand of cancer cells for essential amino acids such as methionine [180] or branched-chain amino acids (BCAAs) [181,182] was previously described. This review addresses only the increased dependencies of cancer cells on vitamins.

3.2 Arginine (L-arginine) and proline

In the TCA cycle, alpha-ketoglutarate is converted into glutamic acid by glutamate dehydrogenase. Glutamic acid is further converted to either glutamine by glutamine synthase, to proline by pyrroline 5-carboxylate reductase 1 and 2 (PYCR1 and 2), or to citrulline (via ornithine), and then arginine, in the urea cycle, with formation of urea from toxic catabolite ammonia in the liver. Thus, arginine, ornithine, and citrulline are all substrates of the urea cycle.

Proline is catabolized into ornithine by proline dehydrogenase 1 (PRODH) and ornithine aminotransferase (OAT), or into glutamic acid by delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH, a.k.a. ALDH4A); ornithine is further converted into glutamic acid by OAT.

Arginine enhances osteogenesis in human mesenchymal stem cells through upregulation of expression of osteogenic transcription factors: RUNX2, DLX5, and OSX (Osterix) [73]. Patients with autosomal recessive mutations in the PYCR2 gene display microcephaly, seizures, facial dysmorphism, developmental delay, and cerebral atrophy [74], and Pycr2^−/− mice exhibit low bone mineral density and decreased grip strength (reported by the IMPC). The catabolism of arginine involves its conversion to glutamic acid by OAT or cycling through the urea cycle. Mutations in OAT cause gyrate atrophy, characterized by progressive retinal atrophy and cataracts, which may cause blindness [75,76].

10.2 Amino acid biosynthesis

Alanine, aspartate, asparagine, glutamate and glutamine are all formed by transamination of pyruvate, oxaloacetate and 2-ketoglutarate, respectively. Proline is formed from glutamate, since γ-glutamyl kinase and 1-pyrroline-5-carboxylate reductase are present. Ornithine can be formed directly from proline by the action of an ornithine cyclodeaminase, or from the proline-pathway intermediate glutamate-semialdehyde. However, ornithine cannot serve as a precursor for arginine because, apart from the urea-cycle enzyme arginase, all other enzymes of this cycle are absent. It is not clear whether serine can be formed from d-3-phosphoglycerate. Although the first enzyme committed to the synthesis of serine: 3-phosphoglycerate dehydrogenase is present, no specific phosphoserine phosphatase homologue was detected. However, serine can be formed from threonine. Serine cannot be used for the synthesis of cysteine and methionine and threonine cannot be synthesised from aspartate via homoserine. Glycine and serine are interconverted in each other by hydroxymethyl transferase and the latter can be formed from threonine as well. A pathway for the synthesis of lysine was not found. Also, the pathways for the synthesis of the branched amino acids valine, leucine, isoleucine, for the aromatic amino acids phenylalanine, tyrosine and tryptophane and nine enzymes necessary for the synthesis of histidine are all missing.

Fulton et al. (1984) carried out studies about the amino acid requirements of N. gruberi when grown in a chemically defined medium. It is satisfying to see that our predictions derived from genomic information are entirely in agreement with these early observations.

In summary, Naegleria has a full capacity for the degradation of all 20 natural l-amino acids it may encounter in prey bacteria and even some d-amino acids present in bacterial cell walls. However it lacks the possibility of synthesising long chain, branched chain and aromatic amino acids, which have to be supplemented in culture medium (Fulton et al., 1984).

5 Proline (pro, pyrrolidine-2-carboxylic acid)

Pro is a non-essential imino acid with important roles in primary metabolism of carbon and nitrogen, protection against osmotic and oxidative stress, protein chaperoning, cellular signaling, apoptosis and adaptation to nutrients. Pro is also necessary for protein synthesis and structure, biosynthesis of amino acids and polyamines, wound healing, ROS scavenging, and immune response [74,75]. It has been described that nutrient formulations comprising Pro exhibit inhibitory activity in distinct PCa cells and xenograft models [76,77]. On the other hand, we have identified elevated urinary Pro levels in patients with diagnosed PCa, whereas virtually no Pro was identified in urinary specimens collected from subjects with no evidence of malignancy [78]. Since PCa cells generally tend to accumulate Pro to scavenge ROS (discussed below), such contradiction could be explained by the minor role of Pro within the nutrient mixture (or marked synergistic effect of mixture components).

Role of miR-23b on ROS production

MiR-23b plays two opposite roles on reactive oxygen species (ROS) production, being involved in the transcriptional regulatory processes that both block or promote ROS, thus causing an anti- or pro-oxidant effect, respectively.