Perspective

Cite This: Biochemistry 2018, 57, 3433−3444 pubs.acs.org/biochemistry

The Proline Cycle As a Potential Cancer Therapy Target John J. Tanner,‡,§ Sarah-Maria Fendt,∥,⊥ and Donald F. Becker*,†

† Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States ‡ § Departments of Biochemistry and Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211, United States ∥ Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology, VIB, Herestraat 49, 3000 Leuven, Belgium ⊥ Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Herestraat 49, 3000 Leuven, Belgium

ABSTRACT: Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ1-pyrroline-5-carboxylate (P5C), which forms L-glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD+- dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline−P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline−P5C cycle has been implicated in supporting ATP production, protein and nucleotide synthesis, anaplerosis, and redox homeostasis in cancer cells. This Perspective details the structures and reaction mechanisms of PRODH and PYCR and the role of the proline−P5C cycle in cancer metabolism. A major challenge in the field is to discover inhibitors that specifically target PRODH and PYCR isoforms for use as tools for studying proline metabolism and the functions of the proline−P5C cycle in cancer. These molecular probes could also serve as lead compounds in cancer drug discovery targeting the proline−P5C cycle.

he pyrrolidine ring makes proline a unique proteogenic water (Figure 1A).1,4,22 The formation of GSAL from ornithine T amino acid with a distinctive role in protein folding and is catalyzed by ornithine δ-amino acid transferase (EC secondary structures. In addition to being required for protein 2.6.1.13). The route to GSAL from glutamate requires biosynthesis, L-proline has critical roles in cellular bioen- γ − − glutamate 5-kinase (G5K; EC 2.7.2.11) and -glutamyl ergetics,1 5 osmoregulation,5,6 stress protection,7 10 cellular phosphate reductase (γ-GPR; EC 1.2.1.41) (Figure 1B). G5K Downloaded via UNIV OF MISSOURI COLUMBIA on August 25, 2018 at 22:08:07 (UTC). signaling processes such as apoptosis,3,11,12 and cancer cell and γ-GPR are separate enzymes in bacteria and lower

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 3,13,14 metabolism. Studies over the last two decades by Phang eukaryotes, whereas in higher organisms, such as plants and and co-workers have established a specialized role for proline in humans, G5K and γ-GPR are fused together in the bifunctional 3,12,13,15−17 cancer metabolism. Further, recent discoveries of the enzyme P5C synthase (P5CS).4,22 The final step of both broad effects of proline metabolism on cancer cell growth and proline biosynthetic routes is the reduction of P5C to proline survival have implicated proline metabolic enzymes as potential catalyzed by NAD(P)H-dependent P5CR (EC 1.5.1.2). In 14,18−21 targets for therapeutic intervention. This Perspective humans, P5CR is known as PYCR, with isoforms PYCR1 and examines the structures and mechanisms of two key proline PYCR2 in the mitochondrion and isoform PYCRL in the Δ1 metabolic enzymes, proline dehydrogenase (PRODH) and - cytosol. pyrroline-5-carboxylate (P5C) reductase (P5CR, a.k.a. PYCR), Breakdown of proline occurs strictly in the mitochondria, and their role in cancer metabolism. The potential for designing with PRODH and GSAL dehydrogenase (GSALDH) localized inhibitors of PRODH and P5CR for cancer therapeutics is also at the inner mitochondrial membrane and matrix, respectively discussed. ■ PROLINE METABOLISM Special Issue: Current Topics in Mechanistic Enzymology Proline is synthesized from ornithine or glutamate, with both Received: February 21, 2018 precursors leading to L-glutamate-γ-semialdehyde (GSAL), an Revised: April 10, 2018 intermediate that spontaneously cyclizes to P5C with loss of Published: April 12, 2018

© 2018 American Chemical Society 3433 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

Figure 1. Reactions and enzymes of proline metabolism in higher organisms. (A) Summary of proline catabolism and biosynthesis. The gray oval represents the mitochondrion. (B) The reactions of proline biosynthesis from glutamate. (C) The reactions of proline catabolism. Abbreviations that are not listed in the text: ORN, ornithine; OAT, ornithine δ-aminotransferase; CAC, citric acid cycle; αKG, α-ketoglutarate; GDH, glutamate dehydrogenase; PPP, pentose phosphate pathway. P5CS is a bifunctional enzyme with G5K and γ-GPR fused together. Adapted with permission from ref 4. Copyright 2017 Mary Ann Liebert, Inc.

(Figure 1A). PRODH (EC 1.5.5.2) performs the first step by bi-bi-ordered mechanism with NAD+ binding and NADH generating P5C, which upon non-enzymatic hydrolysis forms product release being the first and last steps of the reaction, GSAL (Figure 1C). PRODH is a flavin-containing enzyme that respectively.25 The products of the GSALDH reaction, couples the oxidation of proline with reduction of membrane- glutamate and NADH, contribute to energy and nitrogen bound ubiquinone or Coenzyme Q. Humans have two genes metabolism in the cell. NADH feeds electron equivalents to the annotated as PRODH: PRODH1 (chromosome 22q11.21; respiratory chain via mitochondrial complex I, whereas α NCBI Accession NM_016335) and PRODH2 (chromosome glutamate is converted to -ketoglutarate by glutamate fi dehydrogenase, thereby providing carbon to the tricarboxylic 19q13.12; NCBI Accession NM_021232). De ciencies in 1,26,27 PRODH1 manifest in type-I hyperprolinemia,23 whereas the acid cycle (TCA) and ammonia for recycling of nitrogen. loss of PRODH2 activity leads to hydroxyprolinemia,24 consistent with PRODH2 catalyzing the oxidation of trans-4- ■ PRODH STRUCTURE AND MECHANISM 1 hydroxy-L-proline to Δ -pyrroline-3-OH-5-carboxylate (3-OH- Kinetics of Proline Oxidation. Using rat mitochondria, P5C). Johnson and Strecker28 showed that proline is oxidized to P5C + GSALDH (EC 1.2.1.88) catalyzes the NAD -dependent in a reaction that is linked to the respiratory chain. A Km of 2.3 oxidation of GSAL to form glutamate (Figure 1C). Structural mM proline was later determined with a solubilized enzyme and kinetic analysis of human GSALDH shows that it follows a preparation from rat liver mitochondria.29 The first character-

3434 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective ization of a purified PRODH was the native proline utilization A (PutA) enzyme from Escherichia coli. PutA is a bacterial bifunctional enzyme in which PRODH and GSALDH activities are combined into a single polypeptide.30 In some bacteria, such as E. coli, PutAs also have an N-terminal DNA binding domain and regulate transcription of genes putP (proline transporter) and putA.30 PutA from E. coli (EcPutA) was found to be a dimer and to require FAD and electron acceptors for PRODH activity.31 Steady-state kinetic assays of EcPutA have shown that the PRODH reaction follows a two-site ping-pong mechanism,32 similar to that previously observed for the PRODH enzyme from Saccharomyces cerevisiae (Put1p).33 Reduction of the flavin cofactor by proline (reductive half-reaction) is followed by oxidation of reduced flavin by ubiquinone in the membrane 32,33 (oxidative half-reaction). EcPutA PRODH exhibits a kcat of −1 5.2 s for the overall reaction with Km values of 42 mM proline μ 32 −1 and 112 M CoQ1. Microscopic rate constants of 27.5 s for proline reduction of the FAD cofactor and 5.4 s−1 for oxidation fl of reduced FAD by CoQ1 were determined by stopped- ow kinetic measurements, indicating that the oxidative half-reaction is rate-limiting for the overall reaction.34 In a study by Serrano and Blanchard35 on monofunctional PRODH from Mycobacte- rium tuberculosis (MtbPRODH), primary kinetic isotope effects Figure 2. Proposed PRODH mechanism and inhibitors. (A) A conserved active-site Lys residue (Lys234 in human PRODH1) is on V/Km(pro) and V gave evidence that hydride transfer from proposed to deprotonate the proline amine. A stepwise hydride the proline C5 to the N5 of FAD is rate-limiting for the transfer from the C5 of proline to the N5 of FAD then generates P5C reductive half-reaction. Similar to EcPutA PRODH, the and FADH2. (B) Competitive reversible inhibitors and mechanism- oxidative half-reaction was concluded to be rate-limiting for based inhibitors of PRODH. Structures are known for PutAs non- 35 40 41 the overall MtbPRODH reaction. covalently inhibited by L-THFA (PDB IDs 1TIW, 4NMA, 5KF6, 42 39 41 Serrano and Blanchard also showed that a highly a conserved and 5KF7 ) and L-lactate (PDB IDs 4O8A and 4NMB ) and 43 41 Lys residue (Lys110 in MtbPRODH; Lys329 in EcPutA; covalently inactivated by NPPG (PDB IDs 3ITG and 4NME ). Lys234 in human PRODH1) functions as a general base and is critical for catalysis.35 Figure 2A shows a proposed mechanism in which the conserved Lys residue first abstracts an amino could block the binding of 4-hydroxyproline.38 Interestingly, proton from proline. The ensuing hydride transfer from the C5 Tyr540 is conserved in PRODH1 (Tyr548), whereas the − of proline to the N5 of FAD is then shown as a stepwise corresponding residue in PRODH2 is Ser485.38 40 Wild-type 35 −1 −1 transfer as proposed by Serrano and Blanchard. EcPutA-PRODH has kcat/Km values of 492 M s for proline Characterization of purified human recombinant PRODH136 and 3 M−1 s−1 for 4-hydroxyproline.38 An EcPutA-PRODH 37 fi −1 −1 and PRODH2 enzymes, which were signi cantly truncated to Tyr540Ser mutant exhibited kcat/Km values of 11 M s for 4- achieve solubility in E. coli, have confirmed they are flavin- hydroxyproline and 79 M−1 s−1 for proline.38 Replacing Tyr540 dependent enzymes that catalyze the oxidation of proline and with Ser increased the activity with 4-hydroxyproline, but the trans-4-hydroxy-L-proline, respectively. PRODH1 and preference was still for proline, although the activity was >6- PRODH2 are both mitochondrial enzymes and share 45% fold lower.38 38 amino acid sequence identity. Using CoQ1, PRODH2 was PRODHs are also highly selective for the five-membered ring −1 −1 44 reported to have a 12-fold higher kcat/Km (0.93 M s ) with of proline. Studies of EcPutA-PRODH and human 37 trans-4-hydroxy-L-proline relative to L-proline. The product of PRODH136 have found that pipecolate, a six-membered proline the PRODH2 reaction with 4-hydroxy-L-proline is 3-OH-P5C, analogue, is neither a substrate nor an inhibitor. Thus, the which undergoes non-enzymatic hydrolysis to 4-hydroxygluta- upper limit of ring size for potential inhibitor molecules is mate-γ-semialdehyde.38 GSALDH is then able to convert 4- considered to be five. hydroxyglutamate-γ-semialdehyde to 4-hydroxyglutamate.37 PRODH Structure. Currently no structures of PRODH Because 4-hydroxyglutamate feeds glyoxylate production and enzymes from eukaryotes are available. This is largely due to ultimately oxalate, inhibition of PRODH2 has been suggested the fact that eukaryotic PRODH is an inner mitochondrial as a treatment for primary hyperoxaluria in patients with protein, and its expression as a soluble protein in E. coli has disorders in glyoxylate metabolism.37 been problematic. In contrast, the structures of bacterial The substrate selectivity of PRODH1 and PRODH2 was PRODHs have been extensively characterized by high- explored by site-directed mutagenesis of the PRODH domain resolution crystallography, and these structures provide a in EcPutA, which, similar to PRODH1, utilizes 4-hydroxypro- reliable template for modeling of human PRODH (Figure 3A). line poorly as a substrate.31 X-ray crystal structures (2.0 Å The first high-resolution PRODH crystal structure was of the resolution) of the PRODH domain from EcPutA in complex PRODH domain from EcPutA.39 The structure revealed that βα with the proline analogue L-tetrahydro-2-furoic acid (L-THFA) PRODH is a ( )8-barrel with a non-covalently bound FAD were examined to identify active-site residues critical for cofactor. Conservation of the PRODH (βα) -barrel was 8 − substrate recognition.39,40 In EcPutA, Tyr540 was found to confirmed by several structures of full-length PutAs.41 43,47,48 be in close proximity to the C4 of proline, indicating that it The structures of stand-alone PRODH enzymes from Thermus

3435 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

Figure 3. Homology model of residues 121−579 of human PRODH1 made with SWISS-MODEL using the default parameters.45 The template 42 βα chosen by SWISS-MODEL was PDB ID 5KF6. (A) The PRODH ( )8-barrel fold. FAD is colored yellow. The proline analogue L-THFA is colored cyan. The α8 and α5a helices are highlighted in red and orange, respectively. The gray ovals in the right-hand panel indicate residues omitted from the model because of high uncertainty. (B) Close-up views of the predicted active site of human PRODH1. FAD is colored yellow, and L- THFA is colored cyan. βα thermophilus and Deinococcus radiodurans also feature a ( )8- Crystal structures of PutAs in complex with L-lactate and the 49,50 barrel. proline analogue L-THFA have revealed the role of active-site Homology modeling using the most recent update of the residues in substrate binding and conformational dynamics,4,30 βα PDB predicts that the ( )8-barrel fold is present within and all of these residues are predicted to be in the active site of residues 121−579 of human PRODH1 (Figure 3A). In human PRODH (Figure 3B). The conserved sequence motif addition, the modeling predicts two large inserts of uncertain YXXRRXXE on α8 of the barrel is a key element in the structure corresponding to residues 150−205 and 241−349. PRODH active site. The two Arg residues of the motif βα The ( )8-barrel fold and inserts are consistently predicted by (Arg563-Arg564 in human PRODH1) ion-pair with the proline multiple homology modeling servers, such as SWISS- carboxylate (Figure 3B) and are necessary for proline MODEL45 and Phyre2.46 Human PRODH2 is also predicted binding,4,30 while the Tyr residue of the motif (Tyr560) βα to contain the PRODH ( )8-barrel fold; however, it appears packs against the ring of the proline (Figure 3B). Leu527 is to have only one inserted region of unknown structure (240− another highly conserved residue that shapes the active site and 290). provides nonpolar interactions with the proline ring. The Glu An important structural difference between the PutA residue of the motif stabilizes the second Arg residue via ion- PRODH domain and stand-alone bacterial PRODHs is the pairing. The first Arg residue of the YXXRRXXE motif plays an insertion of an α-helix between β-strand 5 and α-helix 5 in important role in substrate binding and active-site dynamics by PutA. Homology modeling predicts that this helix (α5a) is forming an ion-pair gate with a glutamate residue located on present in human PRODHs (Figure 3A). In PutAs, the α5a the β1−α1 loopthis interaction is also predicted to be helix contains a nonpolar residue (Trp or Leu) that packs present in human PRODHs (Figure 3B). Some of the other against the adenine of the FAD, which is presumed to be active-site residues that are conserved in bacterial and human important for establishing the correct conformation of the PRODHs are shown in Figure 3B. Structural and mutational cofactor. In human PRODH, this critical residue is predicted to analysis of these residues in different PRODHs have shown − be Leu447 (Figure 3A). The prediction of α5a in human they have important roles in catalytic steps,35,39,51 53 shaping PRODH suggests that PutAs may be preferred over stand-alone the proline binding site,38,39 FAD conformation and redox bacterial PRODHs as a model system for discovering PRODH potential,51,53 and conformational dynamics.51,52,54 The high inhibitors. structural and sequence conservation in the PRODH active site

3436 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective suggests that these residues have similar catalytic roles in the erythrocyte and lens enzymes are inhibited by NADP+ but human PRODH. not by proline, whereas the fibroblast and lymphoblastoid PRODH Inhibition. Consistent with a conserved PRODH enzymes are inhibited by proline but not by NADP+. Moreover, domain structure across species, L-lactate, pyruvate, and the some enzymes are inhibited by ATP, but others are not. 39,44 proline analogue L-THFA are reversible competitive The differences in cofactor preference and product inhibition inhibitors in several bacterial PutAs/PRODHs31,32 and human caused Dougherty, Brandriss, and Valle63 to posit the existence 36,37 PRODHs (Figure 2B). L-THFA has also been used to of multiple forms of PYCR. Indeed, the use of modern selectively inhibit proline metabolism in bacterial10 and molecular cloning and sequencing tools has revealed three 8,14 mammalian cells. L-THFA inhibits EcPutA PRODH with a PYCR genes, known as PYCR1, PYCR2, and PYCR3 (a.k.a. 32 Ki of 1.6 mM, and a Kd of 1.5 mM was estimated for binding PYCRL), which produce a total of nine PYCR enzymes. 36 71 of L-THFA to human PRODH1. L-Azetidine-2-carboxylate, a (Christensen et al. show an amino acid sequence alignment of four-membered-ring proline analogue (Figure 2B), is a weak the nine PYCR isozymes.) competitive inhibitor of EcPutA PRODH activity (Ki =20 Because it is unclear which isoforms were being assayed in 31 mM). Another potential inhibitor is L-3,4-dehydroproline the early studies, it would be useful to have complete (Figure 2B), which was reported to be a competitive inhibitor comparative kinetic analyses of all of the PYCRs using 55 of PRODH activity in liver mitochondria (Ki = 0.16 mM) and recombinant enzymes. However, only a few papers have has been used to inhibit proline catabolism in cell cultures.56,57 reported the kinetic properties of recombinant human PYCR 72 However, L-3,4-dehydroproline is also a substrate for EcPutA- enzymes. De Ingeniis et al. measured the kinetic parameters 58 PRODH, so the efficacy of L-3,4-dehydroproline as a specific of recombinant PYCR1, PYCR2, and PYCRL using P5C and inhibitor of PRODH activity is unclear and requires further NAD(P)H as the substrates. Christensen et al.71 reported investigation. Furthermore, L-3,4-dehydroproline should be values for PYCR1, also using the forward reaction. Meng et al.59 used with caution especially since it is also a substrate for studied the reverse reaction of PYCR1 using thioproline and PYCR76 (in the reverse of the direction shown in Figure 1) and NAD+ as substrates. can be incorporated into newly synthesized proteins and impact The study by De Ingeniis et al.72 is the only one that 60 collagen formation. compared PYCR isoforms. The Km values for P5C are in the Mechanism-based inhibitors for PRODH have also been low millimolar range, while those of NAD(P)H are a few discovered, such as N-propargylglycine (NPPG)61 and 4- hundred micromolar. The highest catalytic efficiency (k /K ) 62 cat m methylene-L-proline. With both compounds, inactivation was observed with PYCR2 and NADH as the cofactor, while proceeds after the enzyme oxidizes the inhibitor, leaving the the lowest efficiency was seen with PYCR1 and NADPH. They flavin reduced. High-resolution structures of a stand-alone also studied product inhibition by proline. PYCR2 is the most 61 41,48,54 ∼ bacterial PRODH and three PutAs have shown that sensitive to inhibition by proline, with an apparent Ki of 0.1 inactivation by NPPG results in a three-carbon covalent link mM. Because this value is within the range of normal plasma between an active-site lysine residue (Lys329 in EcPutA, proline levels in humans (0.05−0.3 mM73), the inhibition of equivalent to Lys234 in human PRODH1) and the FAD N5 PYCR2 by proline has physiological significance. In contrast, atom. The NPPG-inactivated form of PutA has been a useful the weak inhibition of PYCR1 (Ki = 0.6 mM) and PYCRL (Ki model for analyzing conformational changes that occur upon = 8.5 mM) by proline may not be physiologically significant. 41,48,54 proline reduction of the FAD cofactor. PYCR1 may also play a role in lysine catabolism. Struys et In contrast to proline-analogue-based inhibitors, compounds al.74 asked whether PYCR1 accepts Δ1-piperideine-6-carbox- that target the ubiquinone binding site have generally not been ylate (P6C) as a substrate. P6C is the six-membered-ring explored in PutAs/PRODHs, with the exception of atpenin A5. counterpart of P5C and is an intermediate in lysine catabolism. Atpenin A5, which is a ubiquinone analogue, was shown to be P6C exists in equilibrium with the open-chain α-aminoadipate μ an EcPutA-PRODH competitive inhibitor (Kic =97 M) versus semialdehyde, which is the substrate for the lysine catabolic μ CoQ1 and an uncompetitive inhibitor (Kiu = 124 M) with enzyme aldehyde dehydrogenase 7A1 (ALDH7A1). ALDH7A1 32 respect to proline. Considerably more work is needed to catalyzes the NAD+-dependent oxidation of α-aminoadipate explore ubiquinone-based inhibition of PRODHs. semialdehyde to α-aminoadipate.75 Struys et al.74 showed that human PYCR1 reduces P6C to L-pipecolic acid with kinetic ■ PYCR STRUCTURE AND MECHANISM constants similar to those of the natural substrate. These studies Kinetic Studies of PYCR. Early biochemical studies of suggest the intriguing idea that PYCR1 may have more than PYCR were conducted on enzymes purified from a wide range one metabolic function. of tissues and cell lines, such as liver from rat and calf, human Much remains unknown about the biochemical properties of erythrocytes, rat lens, bovine retina, human fibroblasts, and human PYCR. For example, studies are needed to establish the − human lymphoblastoid cell lines.63 70 The kinetic measure- binding order of substrates. The inhibition of the recombinant ments from these studies reveal general trends and hints about enzymes by NAD(P)+ and ATP has not been studied. Only the fi tissue-speci c variations. The Km for P5C is fairly consistent at three major isoforms have been studied; none of isozymes that − μ 0.1 1.0 M, regardless of the cofactor used. The Km for result from alternative splicing have been expressed and NADPH tends to be lower than that for NADH, sometimes by purified. The impact of inherited disease-causing mutations as much as a factor of 10. The fibroblast and lymphoblastoid on the structure and catalytic function of PYCR has yet to be enzymes are exceptions, having nearly equal Km values for the determined. Finally, investigating the role of PYCR1 in lysine two cofactors. The maximum velocity typically is higher with catabolism is an intriguing line of future research. NADH than with NADPH; however, the ratio of V with Structures of PYCR1. High-resolution crystal structures of NADH to V with NADPH varies substantially, from ∼1 for PYCR1 were determined recently.71 These include structures of retina to 10 for erythrocytes. Also, the sensitivity to product the NADPH substrate complex, proline product complex, and a inhibition by proline and NADP+ is tissue-specific. For example, ternary complex with NADPH and the P5C/proline analogue

3437 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

Figure 4. Structure of PYCR1. (A) The fold of PYCR1 as seen in the ternary complex with NADPH and the proline/P5C analogue L-THFA (PDB ID 5UAV). The N-terminal NAD(P)H binding domain is colored according to secondary structure, with β-strands in pink and α-helices in blue. The C-terminal oligomerization domain is colored gray. NADPH appears in gold sticks. L-THFA is shown as cyan sticks. β-strands are labeled 1−8; α- helices are labeled A−M. Helix-disrupting Pro178 is shown. (B) The structure of the dimer. The α-helices of the C-terminal domain are labeled H− M for the gray protomer and H′−M′ for the purple protomer. NADPH and L-THFA are colored gold and cyan, respectively. The arrow represents the twofold axis of the dimer. (C) The PYCR1 pentamer-of-dimers decamer, with each chain colored differently. (D) The NADPH binding site (PDB ID 5UAT). Selected α-helices and β-strands are labeled as in (A). Helix K (in purple) is from the opposite protomer of the dimer. The 77 conserved water molecule of the dinucleotide-binding Rossmann fold is colored green. (E) The active site of the ternary complex. NADPH and L- THFA are colored gold and cyan, respectively. The two protomers of the dimer are colored purple and gray. The A and L α-helices are labeled. (F) Depiction of the open space in the P5C/proline pocket (PDB ID 5UAU). The product proline is shown in cyan spheres. Water molecules are represented by red nonbonded spheres. Center-to-center distances between water molecules are indicated. (A−E) adapted with permission from ref 71. Copyright 2017 American Society for Biochemistry and Molecular Biology.

L-THFA. Low-resolution structures were reported over a dimer, and the cylindrical decamer are also observed in P5CRs decade ago; however, readers are cautioned that the assignment from microorganisms.76 of the active site in the low-resolution structures was The substrate binding sites have been characterized at high incorrect.59 resolution (1.9 Å). NADPH binds in an extended conformation β The fold of PYCR1 has two domains (Figure 4A). The N- at the C-terminal edge of the -sheet of the Rossmann fold terminal domain has the Rossmann fold and binds NAD(P)H. domain (Figure 4D). This is the canonical pose for NAD(P)- (H) bound to the Rossmann fold. Proline (as well as P5C) The C-terminal domain consists of several α-helices and binds in the dimer interface and interacts primarily with the C- functions in oligomerization and substrate binding. Two terminal domains of the dimer (Figure 4B). The hydrophilic protomers combine to form an interlocked dimer mediated parts of proline bind to the αK−αL loop and conserved Thr238 by the C-terminal domains (Figure 4B). The dimers assemble (shown for a proline analogue in Figure 4E). We note that further around a fivefold axis to form a pentamer-of-dimers Thr238 is present in all PYCR isoforms. The nonpolar ring of decamer (Figure 4C). The fold of PYCR1, the interlocking proline contacts the kink between helices H and I. The kink is

3438 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

Figure 5. Proline metabolism in cancer cell proliferation, survival, and metastasis formation. Proline metabolism supports cancer cell proliferation via biomass precursor production (indicated in blue). Proline metabolism supports or inhibits cancer cell survival via ROS (indicated in red). Proline metabolism supports metastasis formation by promoting invasiveness, clonogenicity, and metastatic seeding (indicated in yellow). Green indicates regulators of proline catabolism and biosynthesis. P5C refers to Δ1-pyrroline-5-carboxylate. caused by the presence of Pro178 and disrupts what would be a Evidence for the proline−P5C cycle was first reported by very long α-helix (Figure 4A). We note that Pro178 is replaced Hagedorn and Phang,78 who showed that NADPH could drive by Val in PYCRL. respiratory activity in mammals without stoichiometric The structure of PYCR1 complexed with NADPH and L- consumption of proline. Evidence for a proline−P5C cycle THFA provides a high-resolution (1.85 Å) model of the ternary has also been shown for plants.79 E·S complex (Figure 4E). The nicotinamide of NADPH meets Whether the proline−P5C cycle is truly directional is P5C in the junction between the dimer interface and the complicated by uncertainties about the subcellular locations Rossmann domain. The rings of the two substrates stack in of the PYCR isoforms. If it is directional, then the reductive parallel, positioning the hydride donor atom of NADPH (C4) half-cycle should be catalyzed by PYCRL, which is known to be 3.7 Å from the acceptor atom of P5C. This arrangement is cytosolic.72 However, Fendt and co-workers concluded that consistent with a direct hydride transfer mechanism. The PYCR1, the isoform thought to be localized in mitochondria,72 structure implies the stereochemistry of hydride transfer. had the most significant role in proline−P5C cycling during Because the B-side of the nicotinamide contacts L-THFA, spheroidal growth of breast cancer cells and was responsible for PYCR1 is predicted to catalyze the transfer of the pro-4S sustaining PRODH activity upon proline withdrawal.14 Phang hydrogen to P5C. et al.80 have suggested that PYCR1 may not be truly The crystal structures should be useful for inhibitor design. mitochondrial but could localize with mitochondrial markers One could target either the P5C site or the NADPH site; as a result of association with mitochondrial outer membranes. however, the former may be preferred because NADPH binds Clarification of the subcellular localization of PYCR1 would be to a conserved fold that is present in many enzymes. The helpful in understanding the role of the proline−P5C cycle in structure of PYCR1 complexed with proline shows a pocket normal redox homeostasis and cancer. with available space that could be exploited (Figure 4F). This Regardless of which PYCR is involved, the proline−P5C pocket binds the nicotinamide riboside of NADPH in the cycle has been shown to enhance oxidative phosphorylation, ternary complex. However, in the proline complex, the pocket maintain cytosolic pyridine nucleotide levels, and generate contains five water molecules. A possible strategy could be to reactive oxygen species (ROS) leading to activation of various design proline analogues with functional groups that extend cell signaling pathways.3,14,15,78,79 The maintenance of NADP+ into the open space, displacing the water molecules and in the cytosol is proposed to link proline metabolism to the forming interactions with the amino acid residues that line the pentose phosphate pathway and nucleotide biosynthe- pocket. sis.3,15,78,81 ■ THE PROLINE−P5C CYCLE ■ PROLINE IN CANCER METABOLISM A unique aspect of proline metabolism is the cycling of proline Cancer cells alter their metabolism to increase survival during and P5C to maintain redox homeostasis between the cytosol cellular stress, to undergo uncontrolled proliferation, and to and mitochondria.2,3 As originally conceived, the proline cycle progress toward metastasis formation.82,83 Proline biosynthesis, consists of catabolic and synthetic half-cycles that occur in catabolism, and cycling have been implicated as metabolic different subcellular locations. The catabolic half-cycle is the pathways selectively altered in cancer cells providing ATP, oxidation of proline to P5C catalyzed by PRODH in macromolecules, and redox cofactors.3 In the following, we will mitochondria (Figure 1A). The synthetic half-cycle is the discuss the emerging role of proline metabolism in light of reduction of P5C back to proline catalyzed by PYCR (Figure cancer cell proliferation, survival, and metastasis formation as 1A). If the reductive step occurs outside mitochondria, the net illustrated in Figure 5. effect of the cycle is to transfer reducing equivalents from Cancer cell proliferation depends on biomass production, i.e., cytosolic NADPH into the mitochondrial respiratory chain. the generation of macromolecules such as DNA and protein

3439 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective from amino acids and other metabolites.83 Proline biosynthesis HIF1α stabilization.91 These effects could contribute to the has been shown to fuel protein production, which is needed for antitumor effect of PRODH expression in some cancers.17 cell proliferation. Accordingly, it has been found that c-MYC Controversial to this potential tumor suppression function of and PI3K signaling, which supports cell proliferation, increases proline catabolism is that in a hypoxic environment PRODH the gene expression of P5CS, PYCR1, PYCR2, and expression has been shown to be increased and contribute to PYCRL.13,15 In kidney cancer, proline is a limiting amino the survival of cancer cells by inducing autophagy.92 Thus, this acid for protein synthesis, and the knockout of PYCR1 is suggests that the environment needs to be considered when sufficient to impair in vivo proliferation in these cancers.84 evaluating the role of PRODH for cancer cell survival. Taken Accordingly, PYCR1 is overexpressed in tumors of human non- together, these results indicate that proline biosynthesis small cell lung carcinoma patients, and knockdown of the inhibition targets cancer cell survival while proline catabolism enzyme impairs proliferation in cell lines.19 Moreover, proline inhibition can have context-dependent pro- or antisurvival derived from collagen has been found to support pancreatic effects in cancer cells. cancer cell proliferation.20 The importance of proline for the One of the most lethal capacities of cancer cells is their ability proliferation of pancreatic cancer cells was indicated not only to metastasize to distant organs. During this process, cancer by direct incorporation of proline into protein but also via cells change their metabolism to invade surrounding tissue, proline catabolism resulting in glutamine, glutamate, and survive in the circulation, and colonize distant organs.83,93,94 aspartate production, which in turn are precursors of DNA Recently, it has been shown that proline metabolism supports and/or protein. Finally, proline biosynthesis can also indirectly this metastatic cascade leading to secondary tumors, i.e., support biomass production by generating the redox cofactor metastases. It has been shown that human metastasis tissue NADP+, which increases the activity of the oxidative pentose exhibits upregulated expression of PRODH compared with phosphate pathway, which in turn generates precursors of primary breast tumor tissue.14 Accordingly, it has been found nucleotide biosynthesis15 needed for DNA and RNA that PRODH inhibition impairs metastasis formation in production. Moreover, it was found that in cancer cells different metastatic breast cancer mouse models without expressing c-MYC, knockdown of proline biosynthesis resulted adverse effects on normal cells and tissues with high PRODH in decreased glycolysis and ATP production.15,85 Interestingly, expression.14 This selectivity toward breast cancer cells can be it was recently shown that in isocitrate dehydrogenase 1 explained on the one hand by the observation that (at least in (IDH1) mutant cancer cells, proline biosynthesis is increased vitro) normal mammary epithelial cells display low PRODH compared with wild-type IDH cancer cells, which results in a expression and on the other hand by the finding that PRODH partial decoupling of the electron transport chain from the inhibitor doses required to impair proline catabolism in TCA cycle.86 Accordingly, ATP-coupled oxygen consumption metastasis tissue are lower than those needed to inhibit 14 increased in IDH1 mutant cancer cells upon proline biosyn- PRODH in liver, heart, and brain. Mechanistically, the proline thesis inhibition. It is therefore tempting to speculate that both cycle composed of PRODH and PYCR1 activity allows observations can impact cancer cell proliferation by perturbing metastasizing breast cancer cells to produce ATP at the 14 the cellular redox balance. In summary, proline metabolism can expense of NADPH, fostering metastatic seeding. Addition- support cancer proliferation and therefore constitutes an ally, inhibition of proline biosynthesis has been found in some interesting intervention point to inhibit the growth of certain cancer cells and yeast to result in unresolved endoplasmic 9,85 tumor types. reticulum (ER) stress. In cancer cells, inhibition of PYCR1 Cancer cells have an increased capacity to survive under hampers clonogenicity, which can support the ability of cancer 85 harsh conditions and to evade apoptosis.87 One important cells to initiate new tumors. Consistent with this observation, mechanism to induce apoptosis and decrease cell survival is the it was found that in human breast cancers PYCR1 gene 18 generation of ROS. Strikingly, proline metabolism can expression is correlated with invasiveness, which is an early contribute to ROS scavenging and generation by different event of the metastatic cascade. While some of these effects means. First, proline itself has some antioxidant capacity. Thus, could be alleviated by exogenous proline, the dependence of increasing proline levels by proline supplementation and metastasizing breast cancer cells on PRODH and PYCR1 could overexpression of PYCR1 or decreasing them by over- not be rescued by proline supplementation or recapitulated by expression of the proline catabolism enzyme PRODH increases depletion of exogenous proline. In conclusion, inhibition of or decreases cellular ROS scavenging, respectively.7,88 Second, proline metabolism by targeting the respective enzymes has the PRODH-catalyzed reaction results not only in ATP emerged as an interesting target to interfere with the metastatic production but also ROS generation.12 Therefore, high proline cascade and prevent metastasis formation. catabolism can induce apoptosis and cell senescence, which has been shown to be counteracted by superoxide dismutase ■ CONCLUSIONS expression in colorectal cancer cells11 or antioxidants in Over the past decade, several major advances have been osteosarcoma cells.89 Accordingly, PRODH expression is achieved in understanding the structures and mechanisms of positively regulated by the tumor suppressor p53,12 while the proline metabolism and its specialized role in cancer oncogene c-MYC negatively regulates PRODH expression via metabolism. Structures of bacterial PRODHs with active-site miR23b*.13,16 Moreover, oral cancer overexpressed 1 ligands are available that enable modeling and structure-based (ORAOV1) protein has been suggested to bind to PYCR1, inhibitor design studies of human PRODH1 and PRODH2. resulting in decreased ROS production, presumably through Structures of human PYCR1 in ternary complex with NADPH activation of proline biosynthesis.90 Additionally, proline and a product analogue provide a template for the design of catabolism can result in production of α-ketoglutarate, which novel inhibitors. A major challenge that remains is solving high- is a metabolic substrate of HIF1α prolyl hydroxylases that resolution structures of human PRODH1, PRODH2, PYCR2, targets HIF1α for degradation,17 and hypoxia can activate and PYCRL. Structures of the these enzymes along with proline biosynthesis in liver cancer cells and might support biochemical characterization of the enzyme mechanisms will be

3440 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective important for designing inhibitors specific for PRODH1 over no negative effects on healthy tissue.14 These results show PRODH2 and for isoform-specific inhibitors of PYCRs. promise for tolerance of PRODH inhibition and proline Covalent inactivation of PRODH by targeting the FAD buildup in healthy human cells. The situation for proline cofactor could be a novel approach by combining specificity biosynthesis is more complicated, as not necessarily the of binding and chemical reactivity. Historically pharma has been products per se but the reactions of the synthetic pathway skeptical of covalent inactivators, but opinions are changing.95 are sometimes most critical. This is illustrated by the different Having small molecules with different modes of inhibition phenotypes of PYCR1 and PYCR2 inborn deficiencies. Loss of would be powerful tools for investigating proline metabolism in PYCR1 gene function is associated with autosomal recessive cancer. cutis laxa with clinical features of wrinkled skin, aged Although structure-based inhibitor discovery is now possible, appearance, and connective tissue weakness.98 Mutations in some practical hurdles need to be overcome. One issue is the PYCR2 gene are associated with autosomal recessive whether the enzymes can be generated in sufficient quantity for neurologic disorder and syndrome of postnatal micro- high-throughput screening of compound libraries. Recombinant cephaly.99,100 Decreased mitochondrial function and oxidative PYCR1 can be expressed and purified from E. coli and is stress tolerance have been reported for PYCR1- and PYCR2- amenable to inhibitor screening. Also, fusion-tagged PYCRs depleted human cells.98,99 Knockdown of PYCR1 or PYCR2 with high activity have been reported and may be suitable as has been shown to have little impact on growth rates, whereas well. Screening for inhibitors of human PRODHs will be more knockdown of PYCRL markedly inhibits proliferation.15 challenging because of the lack of a convenient method for Interestingly, the growth of cells with PYCRL knockdown generating recombinant enzyme. A workaround strategy could could not be rescued by exogenous proline.15 Considering be to use PutA PRODHs as surrogates for human PRODH in these aforementioned effects and the potential impact of screening of compound libraries, followed by testing of hit PYCRs on NAD(P)H redox homeostasis and energy compounds for inhibition of PRODH activity in mitochondrial metabolism (see Figure 5), the likelihood of significant side extracts. Additionally, robust assays need to be developed for effects from drugs targeting proline biosynthesis seems greater drug screening. PRODH activity is typically monitored by than that from drugs targeting PRODH or GSALDH. A measuring reduction of an artificial electron acceptor such as concern for both pathways, however, is the potential for cells to dichlorophenolindolphenol or the formation of a chromogenic accumulate P5C, which was recently listed among the top 30 adduct between o-aminobenzaldehyde and P5C. PYCR activity damage-prone endogenous metabolites.101 Thus, adverse can be measured in the forward direction by following the metabolic effects in healthy tissues and organs will need to be decrease in NAD(P)H absorbance, but some laboratories also examined with any drugs targeting proline metabolic enzymes. measure PYCR activity in the reverse direction using 3,4- Hopefully, though, compounds can be designed to have high dehydroproline as a substrate and monitoring NADH selectivity toward cancer cells. formation.59 Understanding the mechanisms of proline metabolic ■ AUTHOR INFORMATION enzymes can be leveraged to design and discover molecular Corresponding Author probes that could be used to study the functions of the *E-mail: [email protected]. Phone: 402-472-9652. Fax: 402- proline−P5C cycle in cancer metabolism and as lead 472-472-7842. compounds for cancer therapy. Because the proline−P5C ORCID cycle has been shown to significantly influence cellular growth John J. Tanner: 0000-0001-8314-113X and death pathways, the potential of this novel cycle in Donald F. Becker: 0000-0002-1350-7201 tumorigenesis and cancer needs to be further explored.3,14,81 Author Contributions More biochemical details of the proline−P5C cycle are needed The manuscript was written through contributions of all to fully understand its role in cancer metabolism. For example, authors. All authors have given approval to the final version of does the proline−P5C cycle depend on a mitochondrion− the manuscript. cytosol shuttle or is it contained within the mitochondrion? If mitochondrial uptake is required, how is P5C imported into the Funding mitochondrion? Also, are PYCR1 and PYCR2 strictly localized This research was supported by NIGMS, National Institutes of in the inner mitochondrial matrix? Does each PYCR have a Health, Grants R01GM061068 and R01GM065546, and by the specialized role in the proline−P5C cycle depending on the University of Nebraska Agricultural Research Division, cellular context and growth conditions? Dissecting the supported in part by funds provided through the Hatch Act. S.-M.F. acknowledges funding from FWO − Odysseus II, FWO individual roles of PYCRs and PRODH in the proline cycle − − as it relates to cancer will be an important area for future Research Grants/Projects, and KU Leuven Methusalem research. Co-Funding. Finally, issues of how proline metabolic enzyme inhibitors Notes may affect metabolism in healthy cells and tissues needs to be The authors declare no competing financial interest. considered. Presently it appears that inhibiting proline catabolism would have less adverse effects than inhibiting ■ ABBREVIATIONS proline biosynthesis. Inborn deficiencies of the proline catabolic ALDH, aldehyde dehydrogenase; GSAL, glutamate-γ-semi- enzymes PRODH1 and GSALDH lead to hyperprolinemia aldehyde; GSALDH, glutamate-γ-semialdehyde dehydrogenase; metabolic disorders,73 with loss of PRODH1 activity linked to G5K, glutamate 5-kinase; γ-GPR, γ-glutamyl phosphate increased susceptibility to schizophrenia.96,97 How harmful it reductase; NAD+, nicotinamide adenine dinucleotide; NPPG, would be to disrupt proline catabolism in healthy tissues during N-propargylglycine; PRODH, proline dehydrogenase; P6C, Δ1- therapeutic treatment is not clear, but treatment of mice with piperideine-6-carboxylate; P5C, Δ1-pyrroline-5-carboxylate; 1 the PRODH inhibitor L-TFHA blocked lung metastases with P5CR or PYCR, Δ -pyrroline-5-carboxylate reductase; P5CS,

3441 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

Δ1-pyrroline-5-carboxylate synthase; 3-OH-P5C, Δ1-pyrroline- inhibits apoptosis in non-small cell lung cancer. Oncol. Lett. 15, 731− 3-OH-5-carboxylate; PutA, proline utilization A; ROS, reactive 740. oxygen species; L-THFA, L-tetrahydro-2-furoic acid (20) Olivares, O., Mayers, J. R., Gouirand, V., Torrence, M. E., Gicquel, T., Borge, L., Lac, S., Roques, J., Lavaut, M.-N., Berthezene,̀ P., Rubis, M., Secq, V., Garcia, S., Moutardier, V., Lombardo, D., ■ REFERENCES Iovanna, J. L., Tomasini, R., Guillaumond, F., Vander Heiden, M. G., (1) Adams, E., and Frank, L. (1980) Metabolism of proline and the and Vasseur, S. (2017) Collagen-derived proline promotes pancreatic hydroxyprolines. Annu. Rev. Biochem. 49, 1005−1061. ductal adenocarcinoma cell survival under nutrient limited conditions. (2) Phang, J. M. (1985) The regulatory functions of proline and Nat. Commun. 8, 16031. pyrroline-5-carboxylic acid. Curr. Top. Cell. Regul. 25,91−132. (21) Craze, M. L., Cheung, H., Jewa, N., Coimbra, N. D. M., Soria, (3) Phang, J. M. (2012) Proline metabolism in cell regulation and D., El-Ansari, R., Aleskandarany, M. A., Wai Cheng, K., Diez- cancer biology: Recent advances and hypotheses. Antioxid. Redox Rodriguez, M., Nolan, C. C., Ellis, I. O., Rakha, E. A., and Green, A. R. Signaling, DOI: 10.1089/ars.2017.7350. (2018) MYC regulation of glutamine-proline regulatory axis is key in (4) Tanner, J. J. (2012) Structural biology of proline catabolic luminal B breast cancer. Br. J. Cancer 118, 258−265. enzymes. Antioxid. Redox Signaling, DOI: 10.1089/ars.2017.7374. (22) Fichman, Y., Gerdes, S. Y., Kovacs, H., Szabados, L., Zilberstein, (5) Christgen, S. L., and Becker, D. F. (2012) Role of proline in A., and Csonka, L. N. (2015) Evolution of proline biosynthesis: pathogen and host interactions. Antioxid. Redox Signaling, enzymology, bioinformatics, genetics, and transcriptional regulation. DOI: 10.1089/ars.2017.7335. Biol. Rev. Camb Philos. Soc. 90, 1065−1099. (6) Wood, J. M. (2011) Bacterial osmoregulation: a paradigm for the (23) Efron, M. L. (1965) Familial hyperprolinemia. report of a study of cellular homeostasis. Annu. Rev. Microbiol. 65, 215−238. second case, associated with congenital renal malformations, hereditary (7) Krishnan, N., Dickman, M. B., and Becker, D. F. (2008) Proline hematuria and mild mental retardation, with demonstration of an modulates the intracellular redox environment and protects mamma- enzyme defect. N. Engl. J. Med. 272, 1243−1254. lian cells against oxidative stress. Free Radical Biol. Med. 44, 671−681. (24) Efron, M. L., Bixby, E. M., and Pryles, C. V. (1965) (8) Natarajan, S. K., Zhu, W., Liang, X., Zhang, L., Demers, A. J., Hydroxyprolinemia. Ii. A rare metabolic disease due to a deficiency Zimmerman, M. C., Simpson, M. A., and Becker, D. F. (2012) Proline of the enzyme ″hydroxyproline oxidase″. N. Engl. J. Med. 272, 1299− dehydrogenase is essential for proline protection against hydrogen 1309. peroxide-induced cell death. Free Radical Biol. Med. 53, 1181−1191. (25) Srivastava, D., Singh, R. K., Moxley, M. A., Henzl, M. T., Becker, (9) Liang, X., Dickman, M. B., and Becker, D. F. (2014) Proline D. F., and Tanner, J. J. (2012) The three-dimensional structural basis biosynthesis is required for endoplasmic reticulum stress tolerance in of type II hyperprolinemia. J. Mol. Biol. 420, 176−189. Saccharomyces cerevisiae. J. Biol. Chem. 289, 27794−27806. (26) Chao, J. R., Knight, K., Engel, A. L., Jankowski, C., Wang, Y., (10) Zhang, L., Alfano, J. R., and Becker, D. F. (2015) Proline Manson, M. A., Gu, H., Djukovic, D., Raftery, D., Hurley, J. B., and Du, metabolism increases katG expression and oxidative stress resistance in J. (2017) Human retinal pigment epithelial cells prefer proline as a Escherichia coli. J. Bacteriol. 197, 431−440. nutrient and transport metabolic intermediates to the retinal side. J. (11) Liu, Y., Borchert, G. L., Donald, S. P., Surazynski, A., Hu, C.-A., Biol. Chem. 292, 12895−12905. Weydert, C. J., Oberley, L. W., and Phang, J. M. (2005) MnSOD (27) Spinelli, J. B., Yoon, H., Ringel, A. E., Jeanfavre, S., Clish, C. B., inhibits proline oxidase-induced apoptosis in colorectal cancer cells. and Haigis, M. C. (2017) Metabolic recycling of ammonia via Carcinogenesis 26, 1335−1342. glutamate dehydrogenase supports breast cancer biomass. Science 358, (12) Donald, S. P., Sun, X.-Y., Hu, C.-A. A., Yu, J., Mei, J. M., Valle, 941−946. D., and Phang, J. M. (2001) Proline oxidase, encoded by p53-induced (28) Johnson, A. B., and Strecker, H. J. (1962) The interconversion gene-6, catalyzes the generation of proline-dependent reactive oxygen of glutamic acid and proline. IV. The oxidation of proline by rat liver species. Cancer Res. 61, 1810. mitochondria. J. Biol. Chem. 237, 1876−1882. (13) Liu, W., Le, A., Hancock, C., Lane, A. N., Dang, C. V., Fan, T. (29) Kowaloff, E. M., Phang, J. M., Granger, A. S., and Downing, S. J. W. M., and Phang, J. M. (2012) Reprogramming of proline and (1977) Regulation of proline oxidase activity by lactate. Proc. Natl. glutamine metabolism contributes to the proliferative and metabolic Acad. Sci. U. S. A. 74, 5368−5371. responses regulated by oncogenic transcription factor c-MYC. Proc. (30) Liu, L. K., Becker, D. F., and Tanner, J. J. (2017) Structure, Natl. Acad. Sci. U. S. A. 109, 8983−8988. function, and mechanism of proline utilization A (PutA). Arch. (14) Elia, I., Broekaert, D., Christen, S., Boon, R., Radaelli, E., Orth, Biochem. Biophys. 632, 142−157. M. F., Verfaillie, C., Grünewald, T. G. P., and Fendt, S.-M. (2017) (31) Scarpulla, R. C., and Soffer, R. L. (1978) Membrane-bound Proline metabolism supports metastasis formation and could be proline dehydrogenase from Escherichia coli. Solubilization, purifica- inhibited to selectively target metastasizing cancer cells. Nat. Commun. tion, and characterization. J. Biol. Chem. 253, 5997−6001. 8, 15267. (32) Moxley, M. A., Tanner, J. J., and Becker, D. F. (2011) Steady- (15) Liu, W., Hancock, C. N., Fischer, J. W., Harman, M., and Phang, state kinetic mechanism of the proline:ubiquinone oxidoreductase J. M. (2015) Proline biosynthesis augments tumor cell growth and activity of proline utilization A (PutA) from Escherichia coli. Arch. aerobic glycolysis: involvement of pyridine nucleotides. Sci. Rep. 5, Biochem. Biophys. 516, 113−120. 17206. (33) Wanduragala, S., Sanyal, N., Liang, X., and Becker, D. F. (2010) (16) Liu, W., Zabirnyk, O., Wang, H., Shiao, Y. H., Nickerson, M. L., Purification and characterization of Put1p from Saccharomyces Khalil, S., Anderson, L. M., Perantoni, A. O., and Phang, J. M. (2010) cerevisiae. Arch. Biochem. Biophys. 498, 136−142. MicroRNA-23b* targets proline oxidase, a mitochondrial tumor (34) Moxley, M. A., and Becker, D. F. (2012) Rapid reaction kinetics suppressor protein in renal cancer. Oncogene 29, 4914−4924. of proline dehydrogenase in the multifunctional proline utilization A (17) Liu, Y., Borchert, G. L., Donald, S. P., Diwan, B. A., Anver, M., protein. Biochemistry 51, 511−520. and Phang, J. M. (2009) Proline oxidase functions as a mitochondrial (35) Serrano, H., and Blanchard, J. S. (2013) Kinetic and isotopic tumor suppressor in human cancers. Cancer Res. 69, 6414. characterization of L-proline dehydrogenase from Mycobacterium (18) Ding, J., Kuo, M.-L., Su, L., Xue, L., Luh, F., Zhang, H., Wang, J., tuberculosis. Biochemistry 52, 5009−5015. Lin, T. G., Zhang, K., Chu, P., Zheng, S., Liu, X., and Yen, Y. (2017) (36) Tallarita, E., Pollegioni, L., Servi, S., and Molla, G. (2012) Human mitochondrial pyrroline-5-carboxylate reductase 1 promotes Expression in Escherichia coli of the catalytic domain of human proline invasiveness and impacts survival in breast cancers. Carcinogenesis 38, oxidase. Protein Expression Purif. 82, 345−351. 519−531. (37) Summitt, C. B., Johnson, L. C., Jonsson, T. J., Parsonage, D., (19) Cai, F., Miao, Y., Liu, C., Wu, T., Shen, S., Su, X., and Shi, Y. Holmes, R. P., and Lowther, W. T. (2015) Proline dehydrogenase 2 (2018) Pyrroline-5-carboxylate reductase 1 promotes proliferation and (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and

3442 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective molecular target for treating primary hyperoxaluria. Biochem. J. 466, (54) Srivastava, D., Zhu, W., Johnson, W. H., Jr., Whitman, C. P., 273−281. Becker, D. F., and Tanner, J. J. (2010) The structure of the proline (38) Ostrander, E. L., Larson, J. D., Schuermann, J. P., and Tanner, J. utilization a proline dehydrogenase domain inactivated by N- J. (2009) A conserved active site tyrosine residue of proline propargylglycine provides insight into conformational changes induced dehydrogenase helps enforce the preference for proline over by substrate binding and flavin reduction. Biochemistry 49, 560−569. hydroxyproline as the substrate. Biochemistry 48, 951−959. (55) Dashman, T., Lewinson, T. M., Felix, A. M., and Schwartz, M. A. (39) Lee, Y. H., Nadaraia, S., Gu, D., Becker, D. F., and Tanner, J. J. (1979) L-3,4-Dehydroproline: hepatic mitochondrial metabolism and (2003) Structure of the proline dehydrogenase domain of the inhibition of proline oxidase. Res. Commun. Chem. Pathol. Pharmacol. multifunctional PutA flavoprotein. Nat. Struct. Biol. 10, 109−114. 24, 143−157. (40) Zhang, M., White, T. A., Schuermann, J. P., Baban, B. A., Becker, (56) Pandhare, J., Dash, S., Jones, B., Villalta, F., and Dash, C. (2015) D. F., and Tanner, J. J. (2004) Structures of the Escherichia coli PutA A novel role of proline oxidase in HIV-1 envelope glycoprotein- proline dehydrogenase domain in complex with competitive inhibitors. induced neuronal autophagy. J. Biol. Chem. 290, 25439−25451. Biochemistry 43, 12539−12548. (57) Hancock, C. N., Liu, W., Alvord, W. G., and Phang, J. M. (2016) (41) Singh, H., Arentson, B. W., Becker, D. F., and Tanner, J. J. Co-regulation of mitochondrial respiration by proline dehydrogenase/ (2014) Structures of the PutA peripheral membrane flavoenzyme oxidase and succinate. Amino Acids 48, 859−872. reveal a dynamic substrate-channeling tunnel and the quinone-binding (58) Wood, J. M. (1981) Genetics of L-proline utilization in − site. Proc. Natl. Acad. Sci. U. S. A. 111, 3389 3394. Escherichia coli. J. Bacteriol. 146, 895−901. (42) Luo, M., Gamage, T. T., Arentson, B. W., Schlasner, K. N., (59) Meng, Z., Lou, Z., Liu, Z., Li, M., Zhao, X., Bartlam, M., and Becker, D. F., and Tanner, J. J. (2016) Structures of proline utilization Rao, Z. (2006) Crystal structure of human pyrroline-5-carboxylate A (PutA) Reveal the fold and functions of the aldehyde dehydrogenase reductase. J. Mol. Biol. 359, 1364−1377. − superfamily domain of unknown function. J. Biol. Chem. 291, 24065 (60) Kerwar, S. S., and Felix, A. M. (1976) Effect of L-3,4- 24075. dehydroproline on collagen synthesis and prolyl hydroxylase activity in (43) Srivastava, D., Schuermann, J. P., White, T. A., Krishnan, N., mammalian cell cultures. J. Biol. Chem. 251, 503−509. Sanyal, N., Hura, G. L., Tan, A., Henzl, M. T., Becker, D. F., and (61) White, T. A., Johnson, W. H., Jr., Whitman, C. P., and Tanner, J. Tanner, J. J. (2010) Crystal structure of the bifunctional proline J. (2008) Structural basis for the inactivation of Thermus thermophilus utilization A flavoenzyme from Bradyrhizobium japonicum. Proc. Natl. − − proline dehydrogenase by N-propargylglycine. Biochemistry 47, 5573 Acad. Sci. U. S. A. 107, 2878 2883. 5580. (44) Zhu, W., Gincherman, Y., Docherty, P., Spilling, C. D., and (62) Tritsch, D., Mawlawi, H., and Biellmann, J. F. (1993) Becker, D. F. (2002) Effects of proline analog binding on the Mechanism-based inhibition of proline dehydrogenase by proline spectroscopic and redox properties of PutA. Arch. Biochem. Biophys. analogues. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1202, 408, 131−136. 77−81. (45) Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The (63) Dougherty, K. M., Brandriss, M. C., and Valle, D. (1992) SWISS-MODEL workspace: a web-based environment for protein Cloning human pyrroline-5-carboxylate reductase cDNA by com- structure homology modelling. Bioinformatics 22, 195−201. plementation in Saccharomyces cerevisiae. J. Biol. Chem. 267, 871−875. (46) Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and (64) Merrill, M. J., Yeh, G. C., and Phang, J. M. (1989) Purified Sternberg, M. J. (2015) The Phyre2 web portal for protein modeling, − human erythrocyte pyrroline-5-carboxylate reductase. Preferential prediction and analysis. Nat. Protoc. 10, 845 858. − (47) Korasick, D. A., Gamage, T. T., Christgen, S., Stiers, K. M., oxidation of NADPH. J. Biol. Chem. 264, 9352 9358. (65) Matsuzawa, T. (1982) Purification and characterization of Beamer, L. J., Henzl, M. T., Becker, D. F., and Tanner, J. J. (2017) pyrroline-5-carboxylate reductase from bovine retina. Biochim. Biophys. Structure and characterization of a class 3B proline utilization A: − Ligand-induced dimerization and importance of the C-terminal Acta, Gen. Subj. 717, 215 219. domain for catalysis. J. Biol. Chem. 292, 9652−9665. (66) Shiono, T., Kador, P. F., and Kinoshita, J. J. (1986) Purification and characterization of rat lens pyrroline-5-carboxylate reductase. (48) Korasick, D. A., Singh, H., Pemberton, T. A., Luo, M., − Dhatwalia, R., and Tanner, J. J. (2017) Biophysical investigation of Biochim. Biophys. Acta, Gen. Subj. 881,72 78. (67) Yeh, G. C., Harris, S. C., and Phang, J. M. (1981) Pyrroline-5- type A PutAs reveals a conserved core oligomeric structure. FEBS J. − 284, 3029−3049. carboxylate reductase in human erythrocytes. J. Clin. Invest. 67, 1042 (49) White, T. A., Krishnan, N., Becker, D. F., and Tanner, J. J. 1046. (2007) Structure and kinetics of monofunctional proline dehydrogen- (68) Lorans, G., and Phang, J. M. (1981) Proline synthesis and redox ase from Thermus thermophilus. J. Biol. Chem. 282, 14316−14327. regulation: differential functions of pyrroline-5-carboxylate reductase (50) Luo, M., Arentson, B. W., Srivastava, D., Becker, D. F., and in human lymphoblastoid cell lines. Biochem. Biophys. Res. Commun. − Tanner, J. J. (2012) Crystal structures and kinetics of monofunctional 101, 1018 1025. proline dehydrogenase provide insight into substrate recognition and (69) Smith, R. J., Downing, S. J., Phang, J. M., Lodato, R. F., and conformational changes associated with flavin reduction and product Aoki, T. T. (1980) Pyrroline-5-carboxylate synthase activity in − release. Biochemistry 51, 10099−10108. mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 77, 5221 5225. (51) Zhang, W., Zhang, M., Zhu, W., Zhou, Y., Wanduragala, S., (70) Peisach, J., and Strecker, H. J. (1962) The interconversion of 1 Rewinkel, D., Tanner, J. J., and Becker, D. F. (2007) Redox-induced glutamic acid and proline. V. The reduction of Δ -pyrroline-5- changes in flavin structure and roles of flavin N(5) and the ribityl 2′- carboxylic acid to proline. J. Biol. Chem. 237, 2255−2260. OH group in regulating PutA–membrane binding. Biochemistry 46, (71) Christensen, E. M., Patel, S. M., Korasick, D. A., Campbell, A. 483−491. C., Krause, K. L., Becker, D. F., and Tanner, J. J. (2017) Resolving the (52) Zhu, W., Haile, A. M., Singh, R. K., Larson, J. D., Smithen, D., cofactor-binding site in the proline biosynthetic enzyme human Chan, J. Y., Tanner, J. J., and Becker, D. F. (2013) Involvement of the pyrroline-5-carboxylate reductase 1. J. Biol. Chem. 292, 7233−7243. beta3-alpha3 loop of the proline dehydrogenase domain in allosteric (72) De Ingeniis, J., Ratnikov, B., Richardson, A. D., Scott, D. A., Aza- regulation of membrane association of proline utilization A. Blanc, P., De, S. K., Kazanov, M., Pellecchia, M., Ronai, Z., Osterman, Biochemistry 52, 4482−4491. A. L., and Smith, J. W. (2012) Functional specialization in proline (53) Moxley, M. A., Zhang, L., Christgen, S., Tanner, J. J., and biosynthesis of melanoma. PLoS One 7, e45190. Becker, D. F. (2017) Identification of a conserved histidine as being (73) Phang, J. M., Hu, C. A., and Valle, D. (2001) Disorders of critical for the catalytic mechanism and functional switching of the proline and hydroxyproline metabolism. In Metabolic and Molecular multifunctional proline utilization A protein. Biochemistry 56, 3078− Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and 3088. Valle, D., Eds.) pp 1821−1838, McGraw-Hill, New York.

3443 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444 Biochemistry Perspective

(74) Struys, E. A., Jansen, E. E., and Salomons, G. S. (2014) Human Xu, G., and Liu, Y. (2018) Global metabolic profiling identifies a pyrroline-5-carboxylate reductase (PYCR1) acts on Δ1-piperideine-6- pivotal role of proline and hydroxyproline metabolism in supporting carboxylate generating L-pipecolic acid. J. Inherited Metab. Dis. 37, hypoxic response in hepatocellular carcinoma. Clin. Cancer Res. 24, 327−332. 474−485. (75) Luo, M., and Tanner, J. J. (2015) Structural basis of substrate (92) Liu, W., Glunde, K., Bhujwalla, Z. M., Raman, V., Sharma, A., recognition by aldehyde dehydrogenase 7A1. Biochemistry 54, 5513− and Phang, J. M. (2012) Proline oxidase promotes tumor cell survival 5522. in hypoxic tumor microenvironments. Cancer Res. 72, 3677−3686. (76) Nocek, B., Chang, C., Li, H., Lezondra, L., Holzle, D., Collart, (93) Rinaldi, G., Rossi, M., and Fendt, S.-M. (2018) Metabolic F., and Joachimiak, A. (2005) Crystal structures of delta1-pyrroline-5- interactions in cancer: Cellular metabolism at the interface between carboxylate reductase from human pathogens Neisseria meningitides the microenvironment, the cancer cell phenotype and the epigenetic and Streptococcus pyogenes. J. Mol. Biol. 354,91−106. landscape. Wiley Interdiscip. Rev.: Syst. Biol. Med. 10, e1397. (77) Bottoms, C. A., Smith, P. E., and Tanner, J. J. (2002) A (94) Fendt, S.-M. (2017) Is there a therapeutic window for structurally conserved water molecule in Rossmann dinucleotide- metabolism-based cancer therapies? Front. Endocrinol. 8, 150. binding domains. Protein Sci. 11, 2125−2137. (95) Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The (78) Hagedorn, C. H., and Phang, J. M. (1986) Catalytic transfer of resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307−317. hydride ions from NADPH to oxygen by the interconversions of (96) Clelland, C. L., Read, L. L., Baraldi, A. N., Bart, C. P., Pappas, C. proline and delta 1-pyrroline-5-carboxylate. Arch. Biochem. Biophys. A., Panek, L. J., Nadrich, R. H., and Clelland, J. D. (2011) Evidence for 248, 166−174. association of hyperprolinemia with schizophrenia and a measure of (79) Miller, G., Honig, A., Stein, H., Suzuki, N., Mittler, R., and clinical outcome. Schizophr Res. 131, 139−145. Zilberstein, A. (2009) Unraveling delta1-pyrroline-5-carboxylate-pro- (97) Zarchi, O., Carmel, M., Avni, C., Attias, J., Frisch, A., line cycle in plants by uncoupled expression of proline oxidation Michaelovsky, E., Patya, M., Green, T., Weinberger, R., Weizman, enzymes. J. Biol. Chem. 284, 26482−26492. A., and Gothelf, D. (2013) Schizophrenia-like neurophysiological (80) Phang, J. M., Liu, W., and Zabirnyk, O. (2010) Proline abnormalities in 22q11.2 deletion syndrome and their association to metabolism and microenvironmental stress. Annu. Rev. Nutr. 30, 441− COMT and PRODH genotypes. J. Psychiatr. Res. 47, 1623−1629. 463. (98) Reversade, B., Escande-Beillard, N., Dimopoulou, A., Fischer, B., (81) Phang, J. M., Liu, W., Hancock, C., and Christian, K. J. (2012) Chng, S. C., Li, Y., Shboul, M., Tham, P. Y., Kayserili, H., Al-Gazali, L., ’ The proline regulatory axis and cancer. Front. Oncol. 2, 60. Shahwan, M., Brancati, F., Lee, H., O Connor, B. D., Schmidt-von (82) Elia, I., Schmieder, R., Christen, S., and Fendt, S.-M. (2015) Kegler, M., Merriman, B., Nelson, S. F., Masri, A., Alkazaleh, F., Organ-specific cancer metabolism and its potential for therapy. Handb. Guerra, D., Ferrari, P., Nanda, A., Rajab, A., Markie, D., Gray, M., Exp. Pharmacol. 233, 321−353. Nelson, J., Grix, A., Sommer, A., Savarirayan, R., Janecke, A. R., (83) Lunt, S. Y., and Fendt, S.-M. (2018) Metabolism − A Steichen, E., Sillence, D., Hausser, I., Budde, B., Nurnberg, G., cornerstone of cancer initiation, progression, immune evasion and Nurnberg, P., Seemann, P., Kunkel, D., Zambruno, G., Dallapiccola, B., treatment response. Curr. Opin Syst. Biol. 8,67−72. Schuelke,M.,Robertson,S.,Hamamy,H.,Wollnik,B.,Van (84) Loayza-Puch, F., Rooijers, K., Buil, L. C. M., Zijlstra, J., F. Oude Maldergem, L., Mundlos, S., and Kornak, U. (2009) Mutations in − Vrielink, J., Lopes, R., Ugalde, A. P., van Breugel, P., Hofland, I., PYCR1 cause cutis laxa with progeroid features. Nat. Genet. 41, 1016 Wesseling, J., van Tellingen, O., Bex, A., and Agami, R. (2016) 1021. Tumour-specific proline vulnerability uncovered by differential (99) Nakayama, T., Al-Maawali, A., El-Quessny, M., Rajab, A., Khalil, ribosome codon reading. Nature 530, 490−494. S., Stoler, J. M., Tan, W. H., Nasir, R., Schmitz-Abe, K., Hill, R. S., (85) Sahu, N., Dela Cruz, D., Gao, M., Sandoval, W., Haverty, P. M., Partlow, J. N., Al-Saffar, M., Servattalab, S., LaCoursiere, C. M., Liu, J., Stephan, J.-P., Haley, B., Classon, M., Hatzivassiliou, G., and Tambunan, D. E., Coulter, M. E., Elhosary, P. C., Gorski, G., Settleman, J. (2016) Proline starvation induces unresolved ER stress Barkovich, A. J., Markianos, K., Poduri, A., and Mochida, G. H. (2015) − Mutations in PYCR2, encoding pyrroline-5-carboxylate reductase 2, and hinders mTORC1-dependent tumorigenesis. Cell Metab. 24, 753 − 761. cause microcephaly and hypomyelination. Am. J. Hum. Genet. 96, 709 (86) Hollinshead, K. E. R., Munford, H., Eales, K. L., Bardella, C., Li, 719. C., Escribano-Gonzalez, C., Thakker, A., Nonnenmacher, Y., Kluckova, (100) Zaki, M. S., Bhat, G., Sultan, T., Issa, M., Jung, H.-J., Dikoglu, K., Jeeves, M., Murren, R., Cuozzo, F., Ye, D., Laurenti, G., Zhu, W., E., Selim, L., Mahmoud, I. G., Abdel-Hamid, M. S., Abdel-Salam, G., Hiller, K., Hodson, D. J., Hua, W., Tomlinson, I. P., Ludwig, C., Mao, Marin-Valencia, I., and Gleeson, J. G. (2016) PYCR2 mutations cause a lethal syndrome of microcephaly and failure to thrive. Ann. Neurol. Y., and Tennant, D. A. (2018) Oncogenic IDH1 mutations promote − enhanced proline synthesis through pycr1 to support the maintenance 80,59 70. of mitochondrial redox homeostasis. Cell Rep. 22, 3107−3114. (101) Lerma-Ortiz, C., Jeffryes, J. G., Cooper, A. J., Niehaus, T. D., (87) Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: Thamm, A. M., Frelin, O., Aunins, T., Fiehn, O., de Crecy-Lagard, V., − Henry, C. S., and Hanson, A. D. (2016) ’Nothing of chemistry The next generation. Cell 144, 646 674. ’ (88) Guo, J. Y., Teng, X., Laddha, S. V., Ma, S., Van Nostrand, S. C., disappears in biology : the Top 30 damage-prone endogenous metabolites. Biochem. Soc. Trans. 44, 961−971. Yang, Y., Khor, S., Chan, C. S., Rabinowitz, J. D., and White, E. (2016) Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes Dev. 30, 1704−1717. (89) Nagano, T., Nakashima, A., Onishi, K., Kawai, K., Awai, Y., Kinugasa, M., Iwasaki, T., Kikkawa, U., and Kamada, S. (2017) Proline dehydrogenase promotes senescence through the generation of reactive oxygen species. J. Cell Sci. 130, 1413. (90) Togashi, Y., Arao, T., Kato, H., Matsumoto, K., Terashima, M., Hayashi, H., de Velasco, M. A., Fujita, Y., Kimura, H., Yasuda, T., Shiozaki, H., and Nishio, K. (2014) Frequent amplification of ORAOV1 gene in esophageal squamous cell cancer promotes an aggressive phenotype via proline metabolism and ROS production. Oncotarget 5, 2962−2973. (91) Tang, L., Zeng, J., Geng, P., Fang, C., Wang, Y., Sun, M., Wang, C., Wang, J., Yin, P., Hu, C., Guo, L., Yu, J., Gao, P., Li, E., Zhuang, Z.,

3444 DOI: 10.1021/acs.biochem.8b00215 Biochemistry 2018, 57, 3433−3444