PYCR2 is one of three human pyrroline-5-carboxylate reductase isozymes (PYCR1, PYCR2, PYCRL) and catalyzes the final, NAD(P)H-dependent step of proline biosynthesis: reduction of (S)-1-pyrroline-5-carboxylate (P5C) to L-proline (EC 1.5.1.2). It is a mitochondrial enzyme that assembles into a homodecamer built from five homodimers, and it acts chiefly in the glutamate-derived route to proline, using the mitochondrial P5C pool generated by P5C synthase. Like its close paralog PYCR1 (84% identical), PYCR2 can use either NADH or NADPH; at physiological substrate concentrations it shows higher specific activity with NADH, while the historically characterized erythrocyte activity has higher affinity for NADPH and is competitively inhibited by NADP+, leading to the proposal that in some cell types the enzyme also contributes to redox balance by regenerating NADP+. Beyond proline supply, PYCR2 supports mitochondrial membrane potential and resistance to oxidative-stress-induced apoptosis. Biallelic loss-of-function variants in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10), an autosomal recessive disorder with postnatal microcephaly, severe psychomotor delay, hypomyelination and reduced cerebral white-matter volume, indicating a non-redundant role for PYCR2 in the developing central nervous system.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004735 pyrroline-5-carboxylate reductase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of the core catalytic activity of the P5C reductase family. Fully consistent with direct human experimental data (IDA PMID:23024808; EXP PMID:2722838, PMID:6894153) and with the UniProt-annotated EC 1.5.1.2 reaction. This is the core molecular function. Supporting Evidence: file:human/PYCR2/PYCR2-uniprot.txt Oxidoreductase that catalyzes the last step in proline biosynthesis, which corresponds to the reduction of pyrroline-5- |
| GO:0055129 L-proline biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assignment of the core biological process. PYCR2 catalyzes the terminal step of proline biosynthesis; directly supported by the human isotope-tracing / silencing study. Core process. Supporting Evidence: file:human/PYCR2/PYCR2-uniprot.txt L-proline biosynthesis; L-proline from L-glutamate 5-semialdehyde: step 1/1 |
| GO:0004735 pyrroline-5-carboxylate reductase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the catalytic activity via ARBA/InterPro/RHEA/EC mapping (EC 1.5.1.2). Correct and consistent with the experimental IDA/EXP and IBA annotations of the same term; accepted as the core molecular function. Supporting Evidence: file:human/PYCR2/PYCR2-uniprot.txt EC=1.5.1.2 {ECO:0000269|PubMed:23024808, ECO:0000269|PubMed:2722838, |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | TOO GENERAL | Summary: Electronic (SubCell keyword) assignment of cytoplasm. UniProt lists both Cytoplasm and Mitochondrion; the cytoplasm/cytosol annotations derive from the historical erythrocyte enzyme preparation (erythrocytes lack mitochondria) whereas isozyme-resolved fractionation and disease work place PYCR2 in mitochondria. Cytoplasm is over-broad relative to the specific mitochondrial localization, so the specific term replaces it. Reason: Cytoplasm is true but too general: in GO it includes the mitochondria, where isozyme-resolved fractionation and the disease work place PYCR2. Mitochondrion states that location directly. This row is a SubCell keyword mapping with no cell-type context, so unlike the erythrocyte EXP row it is subsumed by the accepted mitochondrion row. |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (ARBA/SubCell) assignment of mitochondrion, consistent with the two experimental IDA mitochondrion annotations. This is the core location. Reason: Synthesis row, examined under the ORGANELLES policy (rule 3). The WITH/FROM is ARBA:ARBA00026962 plus UniProtKB-SubCell:SL-0173 (Mitochondrion). The SubCell keyword is UniProt's curated location for PYCR2, which rests on the two whole-organelle IDA papers (PMID:23024808, PMID:25865492); no source in the chain resolved a sub-compartment, so the coarse term has lost nothing. PYCR2 does have the feature the mapping assumes (experimentally shown mitochondrial residence). A sub-compartment is not added: the only positive line is functional (NADH preference and access to P5CS-derived P5C point to the inside of the organelle but do not separate matrix from the matrix face of the inner membrane), and the mature N-terminus is the acetylated Ser-2 of the primary translation product, so there is no cleaved matrix-targeting sequence to argue for matrix import. The whole-organelle claim is what the evidence supports; ACCEPT. Supporting Evidence: file:human/PYCR2/PYCR2-uniprot.txt Mitochondrion {ECO:0000269|PubMed:23024808, file:human/PYCR2/PYCR2-uniprot.txt PROTEIN SEQUENCE OF 2-17, CLEAVAGE OF INITIATOR METHIONINE, ACETYLATION AT |
| GO:0034599 cellular response to oxidative stress | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Electronic (ARBA) assignment of oxidative-stress response, mirroring the experimental IMP (PMID:25865492). A real but secondary/downstream role (PYCR2 loss lowers mitochondrial membrane potential and sensitizes cells to oxidative-stress apoptosis), not the enzyme's core catalytic function. |
| GO:0055129 L-proline biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (ARBA/InterPro/UniPathway) assignment of the proline biosynthetic process. Correct and consistent with the experimental IDA and the IBA for the same term; accepted as the core biological process. |
| GO:0004735 pyrroline-5-carboxylate reductase activity | EXP PMID:2722838 Purified human erythrocyte pyrroline-5-carboxylate reductase... | ACCEPT | Summary: Direct enzymatic characterization of purified human P5C reductase, establishing NAD(P)H-dependent reduction of P5C to proline, cofactor preference (higher NADPH affinity) and competitive NADP+ inhibition. Strong experimental support for the core catalytic activity. Supporting Evidence: PMID:2722838 The affinity for NADPH is 15-fold higher than that for NADH |
| GO:0004735 pyrroline-5-carboxylate reductase activity | EXP PMID:6894153 Pyrroline-5-carboxylate reductase in human erythrocytes. | ACCEPT | Summary: Identification and kinetic characterization of P5C reductase activity in human erythrocytes. Experimental support for the core catalytic activity (the same enzyme also proposed to help generate NADP+ in erythrocytes). Supporting Evidence: PMID:6894153 which converts pyrroline-5-carboxylate to |
| GO:0005737 cytoplasm | EXP PMID:2722838 Purified human erythrocyte pyrroline-5-carboxylate reductase... | KEEP AS NON CORE | Summary: Cytoplasm annotation derived from purification of the enzyme from human erythrocytes (which lack mitochondria), reflecting the cell type assayed rather than the primary localization of PYCR2 in nucleated cells. Isozyme-resolved fractionation (PMID:23024808) and disease work (PMID:25865492) localize PYCR2 to mitochondria; the cytoplasm assignment is therefore not the core location, but the erythrocyte pool is real. Reason: Kept as non-core, and not modified to mitochondrion like the IEA cytoplasm row: this experiment purified the enzyme from erythrocytes, which have no mitochondria, so it cannot support a mitochondrial location. What it does show is a soluble cytoplasmic pool in one specialized cell type, which is true but is not the location of PYCR2 in nucleated cells. Supporting Evidence: PMID:2722838 The affinity for NADPH is 15-fold higher than that for NADH |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6783939 | KEEP AS NON CORE | Summary: Cytosol location asserted by Reactome, consistent with the erythrocyte enzyme (cytosolic) but not with the mitochondrial localization established for PYCR2 in nucleated cells by isozyme-resolved fractionation and disease studies. Kept as non-core; the core location is mitochondrion. |
| GO:0004735 pyrroline-5-carboxylate reductase activity | IDA PMID:23024808 Functional specialization in proline biosynthesis of melanom... | ACCEPT | Summary: Direct assay of recombinant human PYCR2 (isozyme-resolved), measuring P5C-to-proline reduction kinetics with NADH and NADPH. This is the strongest experimental basis for the core molecular function GO:0004735. Supporting Evidence: PMID:23024808 PYCR1 and PYCR2 are strictly associated with mitochondria |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput mitochondrial-proteome study places PYCR2 in the mitochondrion, corroborating the two low-throughput IDA mitochondrion annotations. Supports the core mitochondrial location. Reason: Direct (HTP) evidence. MitoCoP combined subtractive proteomics of crude vs pure mitochondria, spatial fractionation profiling and TOMM40-knockdown importomics; these assign proteins to mitochondria as a whole and do not resolve sub-compartments. The paper also mapped membrane association by sonication and alkaline extraction, but that per-protein data is in a supplementary table not available in the cached text, so it cannot be used to place PYCR2 at the matrix or the inner membrane. Per policy rule 2 the row says what the assay showed; ACCEPT at the organelle level. Supporting Evidence: PMID:34800366 we combined subtractive proteomics, spatial proteomics, mitochondria-specific importomics, and literature/database curation by stringent criteria |
| GO:0005515 protein binding | IPI PMID:24930674 Frequent amplification of ORAOV1 gene in esophageal squamous... | MARK AS OVER ANNOTATED | Summary: IPI capturing the physical interaction of PYCR2 (as "PYCR") with ORAOV1/LTO1 (UniProtKB:Q8WV07), detected by peptide mass fingerprinting in esophageal squamous cell carcinoma. The interaction is real but "protein binding" is an uninformative molecular function and does not describe PYCR2's activity; marked as over-annotation (an IPI, so not removed). Supporting Evidence: PMID:24930674 ORAOV1 bound to |
| GO:0005739 mitochondrion | IDA PMID:23024808 Functional specialization in proline biosynthesis of melanom... | ACCEPT | Summary: Direct cell-fractionation/immunoblot evidence localizing PYCR2 to mitochondria, distinguishing it from cytosolic PYCRL. This is the core subcellular location of PYCR2. Reason: Direct evidence. The assay was a two-way differential-centrifugation split of Lu1205 cells into a mitochondrial pellet and a cytosolic supernatant, immunoblotted with VDAC1 and IKBalpha as markers. It resolves mitochondrion versus cytosol only; no submitochondrial fractionation, protease protection or alkaline extraction was done, so the curator did not stop short of a sub-compartment the paper resolved. The paper's NADH-preference argument is consistent with an internal (matrix-side) location but is not a localization assay. ACCEPT at the organelle level (policy rule 2). Supporting Evidence: PMID:23024808 PYCR1 and PYCR2 are strictly associated with mitochondria PMID:23024808 This was accomplished with Western blotting using standard approaches to fractionate Lu1205 cells into mitochondrial and cytosolic fractions |
| GO:0055129 L-proline biosynthetic process | IDA PMID:23024808 Functional specialization in proline biosynthesis of melanom... | ACCEPT | Summary: Isotope-tracing plus siRNA silencing show PYCR2 acts in the glutamate-derived route of proline biosynthesis, providing direct evidence for the core biological process. Core process. Supporting Evidence: PMID:23024808 silencing of PYCR2 increased the isotopic enrichment ratio (pro/orn), confirming that this enzyme is predominantly involved in the glutamate pathway |
| GO:0005739 mitochondrion | IDA PMID:25865492 Mutations in PYCR2, encoding pyrroline-5-carboxylate reducta... | ACCEPT | Summary: PYCR2 (wild-type and HLD10 mutant proteins) shown to localize to mitochondria in transfected HEK293FT cells. Independent direct support for the core mitochondrial location. Reason: Direct evidence. Localization of transfected wild-type and HLD10-variant PYCR2 to mitochondria in HEK293FT cells is a whole-organelle result; nothing in the cached abstract (full text not cached) indicates a submitochondrial assay, so there is no resolved sub-compartment for the curator to have missed. ACCEPT at the organelle level (policy rule 2). Supporting Evidence: PMID:25865492 both variant proteins retained normal |
| GO:0034599 cellular response to oxidative stress | IMP PMID:25865492 Mutations in PYCR2, encoding pyrroline-5-carboxylate reducta... | KEEP AS NON CORE | Summary: A CRISPR PYCR2-knockout HEK293FT line showed decreased mitochondrial membrane potential and increased susceptibility to oxidative-stress-induced apoptosis, supporting involvement in the oxidative-stress response. This is a real but downstream/secondary role tied to redox and mitochondrial integrity, not PYCR2's core catalytic function; kept as non-core. Supporting Evidence: PMID:25865492 increased susceptibility to apoptosis under |
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Download this section (compressed HTML)Q: What is the physiological cofactor preference of PYCR2 in mitochondria of nucleated cells, and does its NADH/NADPH usage couple proline synthesis to mitochondrial redox state in a tissue-specific manner?
Q: Why is PYCR2 non-redundant with PYCR1 in the developing CNS despite 84% identity and overlapping mitochondrial localization and activity?
Experiment: Structure- and cell-based dissection of the HLD10 R119C and R251C variants to determine how each destabilizes the decamer/dimer interface and reduces enzyme abundance, relating residual catalytic output to disease severity.
Experiment: Metabolic flux analysis (13C-glutamate/ornithine tracing) in PYCR2-null vs PYCR1-null neural cells to quantify each isozyme's contribution to proline supply and NADP+/NADPH balance during myelination.
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