PYCR2 Human

What is the biochemical function of human PYCR2?

Human PYCR2 (Pyrroline-5-carboxylate reductase 2) catalyzes the final step of L-proline biosynthesis by reducing L-Δ1-pyrroline-5-carboxylate (L-P5C) to L-proline using NAD(P)H as the hydride donor. This reaction represents a critical step in cellular proline metabolism and contributes to maintaining appropriate levels of this amino acid. The enzyme follows a sequential binding mechanism where L-P5C binds before NAD(P)H, and NAD(P)+ is released before L-proline in the catalytic cycle. This ordered binding is essential for the enzyme's proper function and efficiency in maintaining proline homeostasis within cells .

How does PYCR2 differ structurally from other PYCR isoforms?

Human PYCR2 shares 84.4% amino acid sequence identity with PYCR1, making them highly similar in structure. Both PYCR1 and PYCR2 are mitochondrial enzymes, whereas PYCR3 is localized in the cytosol. X-ray crystal structures have been reported for both PYCR1 and PYCR2, revealing their structural similarities and differences. The 1.85 Å structure of human PYCR1 in complex with NADPH and L-tetrahydro-2-furoic acid (L-THFA, a proline analog) confirmed that NADPH binds in the Rao-Rossmann dinucleotide binding domain, which is consistent with the general structural architecture observed in plant and bacterial P5C reductases . Despite their high sequence similarity, PYCR1 and PYCR2 display distinct physiological roles, as evidenced by their association with different disorders.

Where is PYCR2 predominantly expressed in human tissues?

PYCR2 is expressed in various human tissues but has notable enrichment in specific contexts. In cancer studies, immunohistochemistry staining of esophageal squamous cell carcinoma (ESCC) tissue sections identified PYCR2 as the most enriched metabolic enzyme in cancerous regions, consistent with increased L-proline levels found in the same tissue regions . While both PYCR1 and PYCR2 are mitochondrial enzymes, their tissue-specific expression patterns differ, which may explain their association with distinct disorders. PYCR1 mutations are more commonly linked to skin disorders, while PYCR2 mutations appear to be more strongly associated with neurological disorders and brain development .

How are PYCR2 mutations linked to neurological disorders?

Mutations in PYCR2 are uniquely linked to genetic hypomyelinating leukodystrophy type 10 with microcephaly (OMIM # 616420). Unlike PYCR1 mutations which generally lead to cutis laxa type IIB (characterized by skin abnormalities), PYCR2 mutations predominantly affect neurological development. Two specific disease-related variants, Arg119Cys (R119C) and Arg251Cys (R251C), were identified in patients with lethal microcephaly and hypomyelination. These mutations were shown to be catalytically impaired compared to the wild-type enzyme . Loss of PYCR2 has been proposed to impair mitochondrial function and disrupt oxidative balance, particularly affecting NAD(P)+ levels. This suggests that PYCR2 not only produces L-proline but also plays a crucial role in redox cycling of NAD(P)H/NAD(P)+, which may explain its importance in neurological development .

What phenotypes are observed in PYCR2-deficient mouse models?

Mice with recessive loss-of-function mutations in Pycr2 display phenotypes consistent with neurological and neuromuscular disorders. These include weight loss, kyphosis (abnormal spinal curvature), and hind-limb clasping. Despite these neurological symptoms, the peripheral nervous system in these mice shows only mild axonal atrophy in peripheral nerves, with generally normal neuromuscular junctions and nerve conduction velocities .

A striking feature in Pycr2 mutant mice is a severe loss of subcutaneous fat, with DEXA analysis revealing a 53% reduction in total body fat. Histological examination of skin sections confirmed the loss of the hypodermal fat layer in these mice. Additionally, aged Pycr2 mutant mice exhibited reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder beyond neurological effects . While these features are reminiscent of cutis laxa-like phenotypes, primary characteristics such as elastin abnormalities were not observed in these mice.

What is the relationship between PYCR2 and cancer development?

PYCR2 has been implicated in several cancer types. In esophageal squamous cell carcinoma (ESCC), PYCR2 was identified as the most enriched metabolic enzyme in cancerous tissues, correlating with increased L-proline levels in these regions. Studies on human melanoma cell lines demonstrated that silencing PYCR2 expression induced tumor cell death, indicating a critical role for this enzyme in cancer cell survival .

In Kaposi's sarcoma, the viral K1 oncoprotein has been shown to bind endogenous PYCR2, effectively commandeering L-proline biosynthesis to increase intracellular L-proline production. This enhanced proline production promotes 3D spheroid culture growth and tumorigenesis. The viral K1 protein enhances PYCR2 enzymatic activity by lowering the Km for L-P5C by 4-fold and reducing product inhibition by L-proline . These findings suggest that PYCR2 may be a potential therapeutic target in certain cancers where its activity is upregulated or hijacked by oncogenic processes.

How is recombinant human PYCR2 typically expressed and purified for biochemical studies?

For biochemical characterization of human PYCR2, the following expression and purification protocol has been successfully employed:

• Cloning: The DNA sequence encoding human PYCR2 wild-type transcript 1 (NCBI RefSeq NM_013328.4) is subcloned into an expression vector (such as pKA8H) with an N-terminal (8x)Histidine tag.

• Mutagenesis: Site-directed mutagenesis can be performed using overlapping mutagenic and non-mutagenic sequence PCR-based reactions to generate disease-relevant variants.

• Expression: The construct is transformed into E. coli BL21(DE3) cells. Cultures are grown at 37°C until reaching OD600 ≈ 0.8–1.0, then the temperature is lowered to 18°C and IPTG (0.4 mM) is added to induce protein expression.

• Solubility Enhancement: After induction for 10 hours, cultures are treated with chloramphenicol (100 μg/ml) for an additional 2 hours to improve protein solubility through enhanced chaperone-assisted folding.

• Purification: PYCR2 proteins are purified by immobilized metal affinity chromatography (IMAC) using Ni-based resins. The proteins are then concentrated by ultrafiltration and further by freeze-dry vacuum lyophilization.

• Buffer Exchange: The concentrated protein is exchanged into a suitable buffer (e.g., 50 mM HEPES, pH 7.5) by microdialysis.

This protocol typically yields 0.3–4 mg of purified PYCR2 per gram of wet cell pellet . The purified protein can then be used for various biochemical and structural studies, including enzyme kinetics, thermal stability assays, and structural analyses.

What are the optimal assay conditions for measuring PYCR2 enzymatic activity?

The enzymatic activity of PYCR2 can be measured by monitoring the oxidation of NADPH or NADH during the reduction of L-P5C to L-proline. The reaction can be followed spectrophotometrically by measuring the decrease in absorbance at 340 nm, which corresponds to NAD(P)H oxidation.

Optimal assay conditions include:

• Buffer: 50 mM HEPES (pH 7.5)

• Temperature: 37°C

• Substrates: L-P5C (physiological substrate) and NAD(P)H

• Cofactors: NAD(P)H (typically at concentrations between 10-200 μM)

• Enzyme concentration: Typically in the nanomolar range

For kinetic analysis, initial velocities are measured at various concentrations of substrates to determine parameters such as Km and kcat. Product inhibition studies using L-proline and NAD(P)+ can be performed to elucidate the binding mechanism .

It's important to note that PYCR2 follows a sequential binding mechanism with L-P5C binding before NAD(P)H, and NAD(P)+ releasing before L-proline. This mechanistic understanding is crucial for designing appropriate assays and interpreting kinetic data.

How can disease-related PYCR2 variants be functionally characterized?

Functional characterization of disease-related PYCR2 variants involves a multi-faceted approach:

• Enzyme Kinetics: Steady-state kinetic parameters (Km, kcat, kcat/Km) should be determined for both substrates (L-P5C and NAD(P)H) to assess catalytic efficiency. For instance, characterization of the R119C and R251C variants revealed that they are catalytically impaired compared to wild-type PYCR2 .

• Thermostability Analysis: Techniques such as differential scanning fluorimetry or circular dichroism spectroscopy can be used to assess protein stability. This can reveal whether mutations affect protein folding or stability.

• Structural Analysis: X-ray crystallography or cryo-electron microscopy can provide insights into how mutations alter protein structure. In silico modeling may also be useful when experimental structures are unavailable.

• Cellular Localization: Immunofluorescence microscopy can determine whether variants affect the mitochondrial localization of PYCR2.

• Complementation Assays: Functional complementation in model organisms, such as yeast lacking the orthologous enzyme (Pro3), can confirm the enzymatic activity of variants. Both PYCR1 and PYCR2 have been shown to complement the loss of Pro3 in yeast .

• Metabolic Profiling: Measuring proline levels and precursors in relevant biological samples (e.g., serum or cell lysates) can reveal the metabolic consequences of PYCR2 mutations.

These approaches collectively provide a comprehensive understanding of how disease-related variants affect PYCR2 function and contribute to pathology.

How do PYCR1 and PYCR2 functionally interact or compensate for each other?

The functional relationship between PYCR1 and PYCR2 is complex and context-dependent. Studies in mice have shown that Pycr1; Pycr2 double mutants are sub-viable and significantly less healthy compared to either single mutant, indicating that these genes have largely redundant functions in proline biosynthesis . This suggests a compensatory relationship where one enzyme can partially substitute for the loss of the other.

Interestingly, some research suggests that in certain tissues like the brain, loss of PYCR2 can lead to a decrease in PYCR1 levels, and vice versa, suggesting that these proteins may stabilize each other rather than compensate for each other's loss . This complex relationship warrants further investigation to fully understand how these enzymes cooperate in different cellular contexts.

What is the role of PYCR2 in redox homeostasis beyond proline synthesis?

PYCR2 appears to play a significant role in cellular redox balance beyond its function in proline synthesis. The enzyme has been proposed to be important for redox cycling of NAD(P)H/NAD(P)+, suggesting a dual role in both amino acid metabolism and maintaining cellular redox homeostasis .

In the context of neurological disorders, loss of PYCR2 has been proposed to impair mitochondrial function and disrupt oxidative balance, particularly affecting NAD(P)+ levels . This disruption in redox homeostasis could contribute to the neurological phenotypes observed in patients with PYCR2 mutations.

What is the significance of proline metabolism in PYCR2-associated diseases?

The role of proline metabolism in PYCR2-associated diseases is not straightforward. Proline levels are not typically reduced in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, suggesting that overt proline deficiency may not be the primary mechanism underlying PYCR2-associated disorders .

The complexity is further increased by the observed alterations in other metabolic pathways in PYCR2-deficient models. For instance, loss of PYCR2 has been linked to an increase in serine hydroxymethyltransferase (SHMT2) and elevated glycine levels in the brain . This suggests that PYCR2 deficiency may trigger compensatory changes in related metabolic pathways, potentially contributing to disease pathology.

How do experimental models of PYCR2 deficiency compare with human disease phenotypes?

Mouse models of PYCR2 deficiency recapitulate several aspects of human PYCR2-associated disorders, particularly hypomyelinating leukodystrophy type 10 (HLD10). Both humans and mice with PYCR2 mutations show neurological abnormalities, though the specific manifestations differ somewhat between species.

The following table compares key phenotypes between human patients and mouse models:

While the mouse models provide valuable insights, they do not perfectly mirror human disease. The differences may relate to species-specific aspects of proline metabolism or developmental processes. These discrepancies highlight the importance of integrating data from multiple experimental systems when studying PYCR2-related disorders .

What technical challenges exist in studying PYCR2 enzymatic activity?

Several technical challenges complicate the study of PYCR2 enzymatic activity:

Addressing these challenges requires optimization of expression systems, purification protocols, and assay conditions, as well as the use of multiple complementary approaches to validate findings .

What therapeutic approaches might be relevant for PYCR2-associated disorders?

Current understanding of PYCR2 function suggests several potential therapeutic approaches for associated disorders:

Development of effective therapies will require a deeper understanding of the mechanisms by which PYCR2 deficiency leads to disease, as well as identification of suitable biomarkers to monitor disease progression and treatment response.

How might integrative multi-omics approaches advance PYCR2 research?

Integrative multi-omics approaches offer powerful tools for advancing PYCR2 research by providing a holistic view of how PYCR2 functions within the broader cellular context:

• Genomics and Transcriptomics: These approaches can identify additional PYCR2 variants associated with disease and reveal how PYCR2 expression is regulated under different conditions or in different tissues.

• Proteomics: Interaction proteomics can identify PYCR2 binding partners and how these interactions are affected by disease-causing mutations. Post-translational modification profiling can reveal regulatory mechanisms.

• Metabolomics: Comprehensive metabolic profiling can uncover how PYCR2 deficiency affects not only proline metabolism but also connected metabolic pathways, providing insights into secondary consequences of PYCR2 dysfunction.

• Single-cell Approaches: Single-cell transcriptomics and proteomics can reveal cell type-specific roles of PYCR2, potentially explaining the tissue-specific phenotypes observed in patients.

• Systems Biology Integration: Computational integration of multi-omics data can generate testable hypotheses about how PYCR2 functions within cellular networks and how its dysfunction leads to disease.

These approaches, combined with traditional biochemical and structural studies, promise to provide a more complete understanding of PYCR2 biology and potentially identify new therapeutic targets for PYCR2-associated disorders.