pycr3 Antibody

What is PYCR3 and what role does it play in cellular metabolism?

PYCR3 (Pyrroline-5-carboxylate reductase 3, also known as PYCRL) is a cytoplasmic enzyme that catalyzes the final step in proline biosynthesis, specifically converting pyrroline-5-carboxylate (P5C) to proline. Unlike its mitochondrial counterparts PYCR1 and PYCR2, PYCR3 is localized in the cytoplasm and is exclusively linked to the conversion of ornithine to proline . The proline biosynthesis pathway is crucial for various cellular processes, including protein synthesis, cellular structure, and redox balance. PYCR3 utilizes NADPH as a cofactor for this conversion, which links it to cellular redox state and the pentose phosphate pathway .

In cancer metabolism, PYCR3 appears to play a significant role. Recent studies have shown that PYCR3 knockdown results in reduced mitochondrial respiration and cell growth retardation in vitro . This suggests PYCR3 influences not only proline availability but also broader aspects of cellular energy metabolism, despite its cytoplasmic rather than mitochondrial localization.

What are the structural differences between PYCR3 and other PYCR isoforms?

PYCR3 differs from other PYCR family members in several key aspects:

The cytoplasmic localization of PYCR3 is particularly significant as it suggests a distinct metabolic role compared to mitochondrial PYCR1/2 . This compartmentalization allows for specialized regulation of proline synthesis from different precursors, with PYCR3 predominantly utilizing ornithine-derived P5C in the cytosol . The differences in cofactor preference also connect each isoform to distinct metabolic pathways - PYCR3's preference for NADPH links it to the pentose phosphate pathway and cellular redox state.

What applications are PYCR3 antibodies commonly used for in research?

PYCR3 antibodies are employed in multiple research applications:

• Western Blotting (WB): Most commercial PYCR3 antibodies are validated for western blot detection, with typical dilutions ranging from 1:1000-1:4000 . Western blotting enables quantification of PYCR3 protein expression in cell lysates and tissue homogenates.

• Immunocytochemistry/Immunofluorescence (ICC/IF): For subcellular localization studies, PYCR3 antibodies can be used at dilutions of approximately 0.25-2 μg/mL . This application is particularly useful for confirming the cytoplasmic localization of PYCR3 in contrast to mitochondrial PYCR1/2.

• Immunohistochemistry (IHC): Several commercial antibodies are validated for IHC applications, allowing assessment of PYCR3 expression in tissue sections .

• Flow Cytometry (FCM): Some PYCR3 antibodies are suitable for flow cytometry, enabling quantitative analysis of expression in cell populations .

• Immunoprecipitation (IP): PYCR3 antibodies can be used for pulling down PYCR3 and its interacting partners, typically at dilutions around 1:50 . This application has been crucial for discovering interactions such as the PYCR3-USP9x relationship .

• ELISA: Quantitative measurement of PYCR3 in experimental samples, with available kits having detection ranges of approximately 0.156-10 ng/mL .

These applications have been essential for advancing our understanding of PYCR3's role in normal physiology and disease states, particularly in cancer research where expression correlates with disease progression .

How can I validate the specificity of a PYCR3 antibody in my experimental system?

Validating PYCR3 antibody specificity requires a systematic approach:

• Positive and negative controls: Use tissues or cells with known PYCR3 expression patterns. Based on published data, MDA-MB-231 cells and mouse kidney tissue are positive controls for PYCR3 expression . Compare these with tissues known to have minimal expression.

• siRNA/shRNA knockdown: Reduce PYCR3 expression with specific siRNAs and confirm reduced signal intensity. This approach was effectively demonstrated in research examining the USP9x-PYCR3 interaction .

• Overexpression studies: Transfect cells with a PYCR3 expression vector and verify increased signal compared to non-transfected controls.

• Multiple antibodies comparison: Use different antibodies targeting distinct PYCR3 epitopes to confirm consistent staining patterns. Commercial antibodies targeting different regions are available .

• Cross-reactivity testing: Test antibody against recombinant PYCR1 and PYCR2 to ensure isoform specificity. This is particularly important given the sequence homology between PYCR family members across species (human PYCR3 shares 85% homology with mouse PYCR3) .

• Subcellular localization verification: PYCR3 is cytoplasmic, while PYCR1/2 are mitochondrial. Co-staining with mitochondrial markers should show PYCR3 signal outside mitochondria .

• Mass spectrometry validation: After immunoprecipitation with the PYCR3 antibody, perform mass spectrometry analysis. In one study, mass spectrometry identified 17 unique PYCR3 peptides covering 43% of the protein sequence, confirming antibody specificity .

This multi-faceted validation approach ensures that observed signals represent genuine PYCR3 detection rather than cross-reactivity or non-specific binding.

What are the optimal conditions for using PYCR3 antibodies in Western blotting?

Based on published protocols and manufacturer recommendations, optimal conditions for PYCR3 Western blotting include:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • Include proteasome inhibitors (e.g., MG132) if studying ubiquitination status

  • Denature samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels (PYCR3 MW is ~29 kDa)

  • Load 20-40 μg of total protein per lane

  • Consider gradient gels for better separation from PYCR1/2 (~33 kDa)

• Transfer conditions:

  • Use PVDF membrane (preferred over nitrocellulose)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking:

  • 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody:

  • Dilution range: 1:1000-1:4000 for most commercial antibodies

  • Some antibodies recommend 0.04-0.4 μg/mL concentration

  • Incubate overnight at 4°C in blocking buffer

  • Expected band size: 29 kDa (PYCR3 specific)

• Washing:

  • 3-5 washes with TBST, 5-10 minutes each

• Secondary antibody:

  • HRP-conjugated anti-rabbit or anti-mouse (depending on primary)

  • Dilution: 1:5000-1:10000

  • Incubate for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) substrate

  • Exposure to X-ray film or use digital imaging system

• Controls:

  • Include positive controls (MDA-MB-231 cells, mouse kidney tissue)

  • Include loading control (β-actin, GAPDH)

  • For ubiquitination studies, include MG132-treated samples

Following these optimized conditions should yield specific detection of PYCR3 at the expected molecular weight of approximately 29 kDa.

How should I optimize PYCR3 antibody dilutions for immunofluorescence studies?

Optimizing PYCR3 antibody dilutions for immunofluorescence requires a systematic approach:

• Initial titration:

  • Start with manufacturer's recommended dilution range (typically 0.25-2 μg/mL for PYCR3)

  • Prepare a series of dilutions (e.g., 0.1, 0.25, 0.5, 1, 2 μg/mL)

  • Test on positive control samples (cells known to express PYCR3, such as MDA-MB-231)

• Fixation optimization:

  • Test different fixation methods: 4% paraformaldehyde (preserves structure), methanol, or acetone (better for some epitopes)

  • PYCR3 is cytoplasmic, so fixation methods preserving cytoplasmic proteins are generally preferred

• Signal-to-noise evaluation matrix:

• Blocking optimization:

  • Use 5-10% normal serum (from the same species as secondary antibody)

  • Add 0.1-0.3% Triton X-100 for permeabilization

  • Test blocking durations (1 hour vs. overnight)

• Controls to include:

  • Primary antibody omission control (detects non-specific secondary binding)

  • Isotype control (detects Fc receptor binding)

  • Blocking peptide control (confirms epitope specificity)

  • siRNA knockdown control (confirms antibody specificity)

  • Double-staining with mitochondrial markers (confirms cytoplasmic localization)

• Counterstaining:

  • Include DAPI for nuclear staining

  • Consider phalloidin for F-actin cytoskeletal staining

The optimal dilution is the one that produces the strongest specific signal with minimal background. Document all optimization parameters for reproducibility and include representative images of controls in publications.

What are the recommended fixation methods for PYCR3 immunohistochemistry?

For optimal PYCR3 immunohistochemistry, different fixation approaches should be considered based on the sample type and research questions:

• Formalin fixation (FFPE tissues):

  • 10% neutral-buffered formalin for 24-48 hours

  • Paraffin embedding following standard protocols

  • Section at 4-5 μm thickness

  • Antigen retrieval is critical: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Consider testing both acidic (citrate) and basic (EDTA, pH 9.0) retrieval buffers

• Fresh-frozen tissue:

  • Snap freeze in liquid nitrogen or isopentane

  • Embed in OCT compound

  • Section at 8-10 μm thickness

  • Fix sections in cold acetone for 10 minutes

  • Air dry for 20 minutes before staining

• Cultured cells:

  • 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.3% Triton X-100 for 10 minutes

  • Alternative: ice-cold methanol for 10 minutes at -20°C (combines fixation and permeabilization)

• Post-fixation steps:

  • Quench endogenous peroxidase with 3% H₂O₂ in methanol (for HRP-based detection)

  • Block endogenous biotin if using biotin-based detection systems

  • Protein block (5% normal serum or commercial blocking solution) for 1 hour

• Antibody incubation:

  • Dilute primary antibody to optimal concentration (titrate as needed)

  • Incubate overnight at 4°C in a humidified chamber

  • Use polymer-based detection systems for enhanced sensitivity

This approach should be optimized for each specific tissue type, with particular attention to antigen retrieval conditions which can significantly affect PYCR3 detection in FFPE samples. Validation using known positive tissues like kidney is essential for confirming successful protocol implementation.

How can I perform co-immunoprecipitation studies with PYCR3 antibodies?

Based on published research involving PYCR3 protein interactions, particularly the PYCR3-USP9x study , here's a detailed co-immunoprecipitation protocol:

• Cell lysis:

  • Harvest cells at 80-90% confluence

  • Wash twice with ice-cold PBS

  • Lyse cells with IP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors)

  • For ubiquitination studies, add 10 μM MG132 (proteasome inhibitor) to cell culture 4-6 hours before lysis

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Transfer supernatant to a new tube

• Antibody binding:

  • Add 2-5 μg of PYCR3 antibody to pre-cleared lysate

  • For IP-grade antibodies, use recommended dilution (e.g., 1:50)

  • Include appropriate controls:

    • IgG isotype control

    • Input control (10% of lysate before IP)

    • Reverse IP with antibodies against suspected interacting proteins (e.g., USP9x)

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add 50 μL Protein A/G beads

  • Incubate 3-4 hours at 4°C with rotation

  • Collect beads by centrifugation (1000 × g, 1 minute)

  • Wash 5 times with washing buffer (lysis buffer with reduced detergent)

• Elution and analysis:

  • Add 50 μL of 2X SDS-PAGE sample buffer

  • Boil for 5 minutes to elute bound proteins

  • Analyze by Western blotting:

    • Probe for PYCR3 to confirm IP success

    • Probe for suspected interacting proteins (e.g., USP9x)

• For ubiquitination studies:

  • After IP, probe Western blots with anti-ubiquitin antibodies

  • Compare ubiquitination levels under different conditions (e.g., with/without USP9x overexpression)

  • Include deubiquitinase inhibitors in lysis buffer

• For mass spectrometry analysis:

  • Elute with non-denaturing buffer if performing MS analysis

  • Process samples according to standard MS protocols

  • In the PYCR3-USP9x study, MS identified 17 unique PYCR3 peptides covering 43% of the protein sequence

This protocol has been demonstrated to successfully identify and characterize PYCR3 protein interactions, including regulatory mechanisms like deubiquitination that affect PYCR3 stability and function.

How does PYCR3 expression correlate with cancer progression in different tumor types?

PYCR3 expression shows significant correlations with cancer progression and patient outcomes across various tumor types:

These findings collectively indicate that PYCR3 expression has significant potential as a prognostic biomarker across multiple cancer types, with particularly strong evidence in lung, kidney, and hematological malignancies.

What is the relationship between PYCR3 and the regulation of proline metabolism in cancer cells?

PYCR3 plays a crucial role in proline metabolism with specific implications for cancer cell biology:

• Metabolic pathway position:

  • PYCR3 catalyzes the final step in proline biosynthesis in the cytoplasm, specifically from ornithine-derived P5C

  • Unlike mitochondrial PYCR1/2 (which primarily use glutamate-derived P5C), PYCR3 is exclusively linked to the ornithine-to-proline conversion pathway

  • This compartmentalization creates metabolic flexibility for cancer cells

• Metabolic adaptations in cancer:

  • Cancer cells often exhibit altered metabolism to support rapid proliferation

  • Hypoxia (common in tumor microenvironments) increases conversion of 13C-glutamine to proline

  • This metabolic shift likely involves all PYCR isoforms, providing cancer cells with multiple routes to synthesize proline

• NADPH utilization:

  • PYCR3 uses NADPH as a cofactor, connecting proline metabolism to cellular redox state

  • This linkage to the pentose phosphate pathway has implications for cancer cells' ability to manage oxidative stress

  • Cancer cells often exhibit increased flux through the pentose phosphate pathway to generate NADPH

• Impact on cell growth and survival:

  • Depletion of PYCR3 results in cell growth retardation in vitro

  • PYCR3 knockdown leads to reduced mitochondrial respiration, indicating a connection between cytoplasmic proline metabolism and mitochondrial function

  • This suggests PYCR3 supports cancer cell growth through both direct (proline synthesis) and indirect (energy metabolism) mechanisms

• Regulation by post-translational modifications:

  • USP9x deubiquitinates PYCR3, promoting its stability

  • This regulatory mechanism suggests that cancer cells may use post-translational modifications to maintain elevated PYCR3 levels

  • Inhibiting this stabilization could be a potential therapeutic approach

Understanding these relationships provides potential therapeutic targets in cancer treatment. Inhibiting PYCR3 could disrupt cancer cell metabolism at multiple levels, affecting not only proline availability but also redox balance and mitochondrial function.

How does PYCR3 interact with deubiquitinating enzymes and what are the implications for cancer metabolism?

Recent research has revealed a critical regulatory relationship between PYCR3 and deubiquitinating enzymes, particularly USP9x, with significant implications for cancer metabolism:

• Identification of the interaction:

  • Co-immunoprecipitation experiments identified USP9x as a direct interaction partner of PYCR3

  • Mass spectrometric analysis detected 17 unique peptides of PYCR3, covering 43% of the protein sequence

  • This interaction was confirmed by immunoblotting

• Functional relationship:

  • USP9x directly deubiquitinates PYCR3, removing ubiquitin tags that would otherwise target it for proteasomal degradation

  • Overexpression of USP9x markedly reduced ubiquitinated PYCR3 levels

  • This deubiquitination prevents PYCR3 from being degraded, increasing its stability and availability

• Impact on PYCR3 stability:

  • Depletion of USP9x (by siRNAs) or its inhibition (with FT709 inhibitor) reduced PYCR3 protein levels

  • Conversely, USP9x overexpression elevated PYCR3 protein levels

  • This regulation occurs at the protein level, as USP9x knockdown did not affect PYCR3 transcription

• Metabolic consequences:

  • Stabilization of PYCR3 promotes proline biosynthesis in cancer cells

  • Enhanced proline synthesis supports:

    • Protein synthesis (proline makes up ~10% of collagen)

    • Redox balance (through the proline cycle)

    • Cell growth and proliferation

• Mitochondrial function connection:

  • Knockdown of PYCR3 resulted in significant reduction of mitochondrial respiration

  • This suggests a link between cytosolic proline metabolism and mitochondrial function

  • The proline cycle may influence electron transport chain activity

• Clinical implications:

  • High expression of PYCR3 correlated with poor survival in cancer patients

  • This suggests that the USP9x-PYCR3 axis could be a prognostic marker

  • Targeting this interaction might offer therapeutic opportunities

This regulatory mechanism represents a novel post-translational control of proline metabolism in cancer cells and connects ubiquitin-proteasome signaling to metabolic regulation. Therapeutic strategies targeting the USP9x-PYCR3 axis could potentially disrupt this stabilization mechanism and impair proline synthesis in cancer cells.

What experimental approaches can be used to study PYCR3's role in the proline cycle and mitochondrial respiration?

Several experimental approaches can be employed to investigate PYCR3's role in the proline cycle and mitochondrial respiration:

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown: Transiently or stably reduce PYCR3 expression

  • CRISPR-Cas9 knockout: Generate complete PYCR3 knockout cell lines

  • Overexpression systems: Express wild-type or mutant PYCR3 to assess gain-of-function effects

  • Inducible expression systems: Control PYCR3 expression temporally

• Mitochondrial respiration analysis:

  • Seahorse XF analyzer: Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

    • Basal respiration

    • ATP-linked respiration

    • Maximal respiratory capacity

    • Spare respiratory capacity

  • This approach has successfully demonstrated that PYCR3 knockdown reduces mitochondrial respiration

• Metabolic tracing studies:

  • Isotope-labeled precursors:

    • ¹³C-glutamine (as used in multiple myeloma studies)

    • ¹³C-ornithine (to specifically trace PYCR3 activity)

  • Mass spectrometry analysis: Track metabolite flux through proline synthesis pathways

  • This approach revealed increased conversion of ¹³C-glutamine to proline under hypoxic conditions in cancer cells

• Enzymatic activity assays:

  • In vitro PYCR3 activity: Measure conversion of P5C to proline

  • NADPH consumption assays: Monitor cofactor utilization

• Protein interaction studies:

  • Co-immunoprecipitation: Identify protein-protein interactions (successful in identifying PYCR3-USP9x interaction)

  • Proximity labeling: BioID or APEX2 to identify proteins in close proximity to PYCR3

• Cellular compartmentalization studies:

  • Subcellular fractionation: Separate cytosolic and mitochondrial fractions

  • Immunofluorescence microscopy: Visualize PYCR3 localization

• Functional consequences assessment:

  • Cell proliferation assays: Measure growth rates under various conditions

  • Cell viability assays: Assess survival with MTT, CellTiter-Glo, etc.

  • Research has demonstrated that PYCR3 depletion results in cell growth retardation

• Stress response experiments:

  • Hypoxia chambers: Study effects under low oxygen (as used in multiple myeloma studies)

  • Oxidative stress inducers: Test PYCR3's role in redox balance

These approaches can be combined to provide a comprehensive understanding of PYCR3's role in cellular metabolism. Published research has already employed several of these methods to demonstrate PYCR3's impact on mitochondrial respiration and cell growth , providing a foundation for further investigations.

Why might I see multiple bands when probing for PYCR3 in Western blots?

Multiple bands in PYCR3 Western blots can occur for several reasons:

• Isoforms and splice variants:

  • Up to 2 different isoforms have been reported for PYCR3

  • Alternative splicing could produce protein variants of different sizes

• Post-translational modifications:

  • Ubiquitination: PYCR3 is regulated by ubiquitination, which can appear as higher molecular weight bands

  • When studying ubiquitination, researchers detected multiple higher molecular weight bands representing poly-ubiquitinated PYCR3

  • Other potential modifications include phosphorylation, acetylation, or methylation

• Protein degradation products:

  • Incomplete protease inhibition during sample preparation

  • Sample storage conditions (freeze-thaw cycles)

• Cross-reactivity with other PYCR family members:

  • PYCR1 (~33 kDa) and PYCR2 (~33 kDa) vs. PYCR3 (~29 kDa)

  • Sequence homology between family members (human PYCR3 shares homology with mouse PYCR3 at 85%)

• Experimental variations based on literature:

  • Some antibodies detect PYCR1 at both 30 and 32 kDa , suggesting similar variation might occur with PYCR3

  • The calculated molecular weight of PYCR3 is 28.7