PYCRL Human

What is PYCRL and what is its primary function in human cells?

PYCRL (Pyrroline-5-carboxylate reductase 3 or Pyrroline-5-carboxylate reductase-like protein) is an enzyme that catalyzes the last step in proline biosynthesis. It specifically catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the hydride donor . While humans have three isoforms of pyrroline-5-carboxylate reductase (PYCR1, PYCR2, and PYCRL), PYCRL is unique in being exclusively linked to the conversion of ornithine to proline, rather than glutamate to proline . This specialized role distinguishes it from its mitochondrial counterparts PYCR1 and PYCR2, which are primarily involved in glutamate-to-proline conversion.

Methodologically, this distinction was established through metabolic fate tracking of 13C-labeled precursors in human melanoma cells, revealing the exclusive association of PYCRL with the ornithine-proline pathway .

How does PYCRL differ structurally and functionally from other PYCR family members?

PYCRL differs from PYCR1 and PYCR2 in several key aspects:

• Structural differences: PYCRL is approximately 40 amino acids shorter at the C-terminus compared to PYCR1 and PYCR2, and shares only about 45% similarity with the other two forms . PYCRL is 274 amino acids in length, while PYCR1 (319aa) and PYCR2 (320aa) are very similar to each other (84% similarity) .

• Subcellular localization: PYCRL is localized in the cytosol, whereas PYCR1 and PYCR2 are localized in the mitochondria .

• Functional specialization: PYCRL is exclusively involved in converting ornithine to proline, while PYCR1 and PYCR2 primarily convert glutamate to proline .

• Enzymatic properties: While all three enzymes catalyze the reduction of P5C to proline, they may have different cofactor preferences and kinetic properties, though the search results do not provide specific details on these differences for PYCRL.

What is the genomic location of PYCRL and how is its expression regulated?

The PYCRL gene is located on chromosome 8q24.3 in humans . Expression regulation of PYCRL varies across different cell types and conditions. In melanocytes, PYCRL is expressed to some degree but shows higher expression in some melanoma cell lines, suggesting potential upregulation in certain cancer contexts .

For experimental quantification of PYCRL expression, researchers use qPCR with specific primers: PYCRL forward 5′-cccagaccctgctgggggacg-3′ and PYCRL reverse 5′-ctccacggcgctcatggtgg-3′, with human cyclophilin A commonly used as a control . Reaction conditions typically involve denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30s, annealing at 56°C for 60s, and extension at 72°C for 30s .

How does PYCRL contribute to cancer metabolism and progression?

PYCRL's role in cancer appears to be context-dependent. In melanoma, PYCRL shows increased expression in some cell lines compared to normal melanocytes, suggesting a potential role in melanoma metabolism . When PYCRL was silenced with siRNA in melanoma cells, researchers observed a reduction in cell proliferation, though smaller than the effect seen with PYCR1 silencing .

In breast cancer, the evidence is less clear. One study noted that PYCRL did not significantly impact the outcome of breast cancer, unlike PYCR1 which was found to promote invasiveness and impact survival . This suggests that different PYCR family members may have distinct roles across cancer types.

Cancer cells often show metabolic reprogramming, favoring de novo synthesis pathways over salvage pathways. In the case of proline metabolism, tumor cells tend to rely on proline biosynthesis rather than salvage mechanisms . PYCRL's specific role in converting ornithine to proline may be particularly important in cancer contexts where this metabolic pathway is upregulated.

What methods are most effective for studying PYCRL function in cellular models?

Studying PYCRL function effectively requires a combination of molecular, biochemical, and metabolic approaches:

• Gene Silencing/Overexpression: siRNA knockdown of PYCRL is effective for studying its function. Researchers have successfully used this approach to examine PYCRL's role in cell viability and metabolism . For overexpression studies, PYCRL can be amplified by PCR (using primers: forward 5′-acacacggatccatggcagctgcgg-3′ and reverse 5′-gtgccactcgagctactttctgctgagctcc-3′) and cloned into appropriate expression vectors, such as pSMT3 with an N-terminal 6xHis_SUMO tag .

• Metabolic Tracing: 13C labeling experiments have been crucial for delineating PYCRL's specific role in proline biosynthesis. By tracking the fate of labeled precursors (glutamate vs. ornithine) to proline after PYCRL silencing, researchers can quantify its contribution to specific metabolic pathways .

• Recombinant Protein Production: For biochemical and structural studies, PYCRL Human Recombinant can be produced in E. coli as a single, non-glycosylated polypeptide chain containing the 274 amino acids of the native protein .

• Enzymatic Assays: Activity assays can be performed using purified recombinant PYCRL with P5C as substrate and NAD(P)H as cofactor, monitoring the oxidation of NAD(P)H spectrophotometrically.

How do mutations in PYCRL affect enzyme function and what are the potential physiological consequences?

While the search results don't specifically mention disease-associated mutations in PYCRL, they do describe disease-related variants in the related enzyme PYCR2, providing insight into how mutations might affect PYCR family enzymes. In PYCR2, disease-related variants (Arg119Cys and Arg251Cys) show significantly impaired catalytic efficiency and, in the case of R251C, a pronounced folding defect .

By analogy, mutations in PYCRL might affect:

• Catalytic Efficiency: Mutations in key catalytic residues could reduce the enzyme's ability to convert P5C to proline, potentially limiting proline availability.

• Protein Stability: Some mutations might affect protein folding and stability, as seen with the R251C variant of PYCR2 .

• Substrate or Cofactor Binding: Mutations in binding sites could alter the enzyme's affinity for P5C or NAD(P)H.

Potential physiological consequences might include:

• Altered cellular proline levels, which could affect protein synthesis and structure

• Changes in cellular redox status, as the proline synthesis pathway is linked to NADPH oxidation

• Impacts on cellular stress responses, as proline serves as a non-enzymatic antioxidant against reactive oxygen species

What are the optimal conditions for expressing and purifying recombinant PYCRL for biochemical studies?

Recombinant PYCRL can be effectively produced in E. coli expression systems. Based on the available information, the following methodology is recommended:

• Expression System: E. coli has been successfully used for PYCRL expression . The gene should be cloned into a suitable vector, such as pSMT3, which allows for the addition of an N-terminal 6xHis_SUMO tag to facilitate purification .

• Gene Amplification: The PYCRL gene can be amplified using specific primers (forward 5′-acacacggatccatggcagctgcgg-3′ and reverse 5′-gtgccactcgagctactttctgctgagctcc-3′) .

• Protein Characteristics: The resulting recombinant protein is a single, non-glycosylated polypeptide chain containing 297 amino acids (covering positions 1-274 of the native sequence) with a His-tag for purification .

• Quality Assessment: The purified protein should have >90% purity and be suitable for techniques such as SDS-PAGE and mass spectrometry .

For researchers requiring ready-made recombinant PYCRL, commercial sources offer human PYCRL with verified purity suitable for biochemical and enzymatic studies .

How can isotopic labeling experiments be designed to distinguish PYCRL's metabolic contributions from other PYCR isoforms?

Isotopic labeling experiments are crucial for distinguishing PYCRL's specific contributions to proline biosynthesis from those of PYCR1 and PYCR2. Based on successful approaches in the literature :

• Experimental Design Principle: Track the fate of 13C-labeled precursors (glutamate and ornithine) to proline in cells with and without PYCRL silencing. Since PYCRL is exclusively linked to the ornithine-proline pathway, changes in isotopic enrichment patterns will reveal its specific contribution.

• Silencing Approach: Use siRNA to specifically target PYCRL. When an enzyme functioning along a pathway contributing the isotopically labeled precursor is silenced, isotopic enrichment in the product (proline) relative to the precursor will decrease.

• Interpretation Framework:

  • If PYCRL primarily utilizes the ornithine pathway (with non-isotopically enriched carbon) and 13C-glutamate is used as a tracer, silencing PYCRL will decrease flux of 12C toward proline, potentially causing an increase in isotopic enrichment from the glutamate pathway.

  • Control experiments should include tracking other metabolites to ensure observed changes in proline labeling are specific to the targeted enzyme and not due to general metabolic effects .

• Validation Approach: Compare results with similar experiments targeting PYCR1 and PYCR2 to confirm the distinct roles of each isoform.

This experimental design has successfully demonstrated that PYCRL is exclusively linked to the ornithine-proline pathway, while PYCR1 and PYCR2 are primarily involved in the glutamate-proline pathway .

What qPCR protocols are most reliable for quantifying PYCRL expression in human tissue samples?

For reliable quantification of PYCRL expression in human tissue samples, the following qPCR protocol has been validated in research settings :

• Primer Selection:

  • PYCRL forward primer: 5′-cccagaccctgctgggggacg-3′

  • PYCRL reverse primer: 5′-ctccacggcgctcatggtgg-3′

• Reference Gene:

  • Human cyclophilin A is recommended as a control for normalization.

• Reaction Conditions:

  • Denaturation: 95°C for 10 minutes

  • Cycling (40 cycles): 95°C for 30 seconds, 56°C for 60 seconds, 72°C for 30 seconds

  • Use SYBRGreenER Universal qPCR Mix or equivalent reagents

  • Run on a qPCR cycler such as MX3000P or similar systems

• Quality Control:

  • Verify the specificity of the products by melting curve analysis

  • Include no-template controls to check for contamination

  • Run technical replicates to ensure reproducibility

• Data Analysis:

  • Normalize PYCRL mRNA levels to the reference gene (cyclophilin A)

  • Use the comparative Ct (2-ΔΔCt) method for relative quantification

  • Validate significant changes with biological replicates

This protocol has been successfully used in studies examining PYCRL expression in melanoma cell lines and can be adapted for various human tissue samples .

How does PYCRL function differ between normal and cancerous tissues?

PYCRL shows distinct expression and functional patterns between normal and cancerous tissues, though research in this area is still developing:

The specific contribution of PYCRL to cancer metabolism appears to be cancer-type dependent and requires further research to fully elucidate its role across different malignancies.

What are the key differences in substrate specificity and enzymatic efficiency between PYCRL and other PYCR isoforms?

While detailed kinetic parameters specifically for PYCRL compared to other PYCR isoforms are not provided in the search results, several key functional differences have been established:

For researchers planning to characterize PYCRL's enzymatic properties in comparison to other PYCR isoforms, a comprehensive kinetic analysis similar to that performed for PYCR2 would be valuable , including determination of:

• Substrate binding order

• Catalytic efficiency with different cofactors (NADH vs. NADPH)

• Product inhibition kinetics

• Thermostability characteristics

What are the unresolved questions regarding PYCRL's role in human metabolism and disease?

Several important questions about PYCRL remain unresolved, presenting opportunities for future research:

• Precise Regulatory Mechanisms:

  • How is PYCRL expression regulated in different tissues and under various physiological conditions?

  • What transcription factors and signaling pathways control PYCRL expression?

• Disease Associations:

  • While mutations in PYCR1 have been linked to cutis laxa , and PYCR2 mutations are associated with microcephaly and hypomyelination , the potential role of PYCRL variants in human diseases remains largely unexplored.

  • Are there specific disorders associated with PYCRL dysfunction?

• Metabolic Integration:

  • How does PYCRL's activity integrate with broader metabolic networks, especially in relation to ornithine metabolism, urea cycle activity, and polyamine synthesis?

  • Does PYCRL function as part of a metabolic complex or interact with other enzymes?

• Therapeutic Potential:

  • Could PYCRL be a viable therapeutic target in specific disease contexts?

  • Given its role in cell proliferation , could PYCRL inhibition be explored as a cancer treatment strategy?

• Structural Biology:

  • Detailed structural information about PYCRL, including substrate binding sites and catalytic mechanism, would enhance understanding of its function.

  • How does PYCRL's structure differ from PYCR1 and PYCR2, particularly in relation to its substrate specificity?

How might PYCRL be targeted therapeutically in diseases where proline metabolism is dysregulated?

Targeting PYCRL therapeutically presents both opportunities and challenges for diseases involving dysregulated proline metabolism:

• Target Validation Approaches:

  • Genetic knockdown studies in disease models to confirm PYCRL's role

  • Metabolic profiling to identify contexts where the ornithine-proline pathway is especially important

  • Comparative analysis with existing PYCR1/2 inhibition studies to determine unique effects of PYCRL targeting

• Potential Therapeutic Strategies:

  • Small molecule inhibitors designed specifically for PYCRL's active site

  • Allosteric modulators that could alter PYCRL's activity without completely inhibiting it

  • Targeted protein degradation approaches using proteolysis-targeting chimeras (PROTACs)

• Disease Contexts to Consider:

  • Cancer: In melanoma cells, PYCRL silencing reduced cell proliferation , suggesting potential anticancer applications

  • Metabolic disorders: Conditions involving ornithine accumulation might benefit from PYCRL modulation

  • Fibrotic diseases: Given proline's importance in collagen synthesis, PYCRL inhibition might affect fibrosis progression

• Combination Therapy Potential:

  • In cancer contexts, PYCRL inhibition could potentially be combined with existing therapies

  • Similar to how chemotherapy showed improved effects in breast cancer patients with low PYCR1 expression , PYCRL targeting might sensitize certain tumors to standard treatments

• Selectivity Considerations:

  • Developing inhibitors that specifically target PYCRL without affecting PYCR1/2 would be crucial

  • The structural differences between PYCRL and other family members (only 45% similarity) provide a basis for selective targeting

The development of therapeutic approaches targeting PYCRL would benefit from more detailed enzymatic characterization and structural information, as well as broader investigation of its role in various disease contexts.