PGPEP1 Human

What is PGPEP1 and what is its primary function?

PGPEP1 (Pyroglutamyl-peptidase I) is a cytosolic cysteine peptidase belonging to the peptidase C15 family of proteins. It functions as an omega peptidase that specifically removes pyroglutamyl residues from amino termini of peptides and proteins . This enzyme requires a thiol-reducing agent for activity .

The primary biological role of PGPEP1 involves the inactivation of biologically active peptides possessing an amino terminal pyroglutamyl group . Key substrates include several important neuropeptides and hormones such as neurotensin, luteinizing hormone releasing hormone (LHRH), and thyrotropin-releasing hormone (TRH) .

What is the molecular structure and cellular localization of PGPEP1?

Human PGPEP1 is a 209-amino acid protein (Met1-His209) with a predicted molecular mass of 24 kDa , though it typically appears between 23-26 kDa on SDS-PAGE under reducing conditions . The protein is predominantly cytosolic and expressed in most cell types .

Western blot analyses have detected PGPEP1 in various human cells and tissues including:

• LNCaP (prostate cancer cell line)

• JAR (choriocarcinoma cell line)

• NTera-2 (testicular embryonic carcinoma cell line)

• Human brain cortex tissue

Human and mouse PGPEP1 share 95% amino acid sequence identity, indicating strong evolutionary conservation .

How is PGPEP1 genetically encoded and regulated?

PGPEP1 is encoded by the PGPEP1 gene (Entrez Gene ID: 54858) in humans . Synonyms for this gene include PAP-I, PGI, PGP, PGP-I, PGPI, and Pcp .

The regulation of PGPEP1 appears to be tissue-specific and context-dependent. While detailed transcriptional regulatory mechanisms aren't fully characterized in the available literature, expression levels appear to be modulated in certain disease states. For instance, PGPEP1 gene expression is reportedly downregulated in colorectal cancer , suggesting disease-specific transcriptional regulation.

Fluorogenic substrate assay:

The gold standard for PGPEP1 activity measurement is monitoring the hydrolysis of pyroglutamyl-AMC (pyroglutamyl-7-amino-4-methylcoumarin) . Under optimal conditions including:

• Buffer containing DTT as reducing agent

• Controlled temperature and pH

• The specific activity exceeds 9,500 pmol/min/μg

Western blot analysis (for protein levels):

For detection and semi-quantitative analysis of PGPEP1 protein:

• Use PVDF membrane probed with specific antibodies (e.g., Sheep Anti-Human PGPEP1 Antibody)

• Apply 1 μg/mL of primary antibody followed by HRP-conjugated secondary antibody

• Expected band: approximately 23 kDa under reducing conditions

• Recommended positive controls: LNCaP, JAR, NTera-2 cell lines, human brain tissue

Recombinant protein handling considerations:

• Storage: -20 to -70°C for long-term stability

• After reconstitution: 2-8°C for up to 1 month; -20 to -70°C for up to 6 months

• Avoid repeated freeze-thaw cycles

What is the relationship between PGPEP1 and colorectal cancer?

The relationship between PGPEP1 and colorectal cancer (CRC) involves complex patterns with the gene, protein, and its circular RNA form:

Clinical correlations demonstrate that high circPGPEP1 expression in CRC patients is associated with:

• Lymph node metastasis

• Poor survival prognosis (per Kaplan-Meier analysis)

Functional studies show that circPGPEP1 silencing inhibits CRC cell proliferation, suggesting its potential role in promoting cancer progression .

What is circPGPEP1 and how is it related to the PGPEP1 gene?

circPGPEP1 is a circular RNA derived from the PGPEP1 gene locus. Key characteristics include:

• Structure and stability: circPGPEP1 demonstrates remarkable stability compared to linear mRNAs, with a significantly longer half-life than GAPDH mRNA and resistance to RNase R digestion .

• Expression pattern: While the PGPEP1 gene itself appears downregulated in colorectal cancer, circPGPEP1 shows significant upregulation in CRC tissues and cell lines compared to normal controls .

• Functional significance: Experimental evidence indicates that circPGPEP1 plays a role in promoting CRC cell proliferation, as silencing circPGPEP1 effectively inhibits this process .

• Clinical relevance: High circPGPEP1 expression correlates with lymph node metastasis and poor survival outcomes in CRC patients, suggesting potential utility as a prognostic biomarker .

This inverse relationship between PGPEP1 gene expression and circPGPEP1 levels suggests possibly antagonistic functions in cancer biology, representing an important area for further investigation.

How can researchers effectively express and purify PGPEP1 for functional studies?

For optimal expression and purification of human PGPEP1:

Expression systems:

• E. coli-based expression has been successfully used for human PGPEP1 (Met1-His209)

• Recombinant human PGPEP1 can be produced with C-terminal tags (e.g., 6-His tag) to facilitate purification

Purification approach:

• Affinity chromatography yields >90% purity as verified by SDS-PAGE under reducing conditions

• Expected molecular mass: 24-26 kDa on SDS-PAGE

• Quality control should include N-terminal sequencing to confirm Met1 and endotoxin testing (<1.0 EU per 1 μg)

Formulation considerations:

• Optimal buffer components: Tris, NaCl, Glycerol, and DTT

• DTT is critical as a reducing agent to maintain enzymatic activity

• Filter sterilization (0.2 μm) for applications requiring sterility

Functional validation:

• Activity verification through pyroglutamyl-AMC hydrolysis assay

• Specific activity threshold: >9,500 pmol/min/μg under standardized conditions

Gene expression manipulation:

• Overexpression strategies:

  • Mammalian expression vectors containing PGPEP1 cDNA (e.g., pCMV6-XL6 vector)

  • Transfection optimization based on cell type (lipid-based methods, electroporation)

  • Validation of expression by Western blot and activity assays

• Silencing approaches:

  • siRNA/shRNA targeting specific PGPEP1 sequences

  • CRISPR-Cas9 for complete knockout studies

  • For circPGPEP1 studies, specific targeting of back-splice junctions is required

Functional assessment:

• Enzyme activity: Measure substrate processing using synthetic or natural substrates

• Cellular phenotypes: Proliferation, migration, differentiation

• Peptide metabolism: LC-MS/MS analysis of pyroglutamyl peptide levels

• Cancer-specific assays: When studying oncological contexts, assess:

  • Cell proliferation rates

  • Colony formation capacity

  • Migration/invasion potential

  • Xenograft tumor growth in animal models

The opposite effects observed between PGPEP1 and circPGPEP1 in colorectal cancer suggest investigating both in parallel may yield important insights into their potentially antagonistic functions .

What are the critical factors affecting PGPEP1 enzyme activity measurements?

Several factors critically influence PGPEP1 activity measurements:

• Reducing environment: PGPEP1 absolutely requires thiol-reducing agents (e.g., DTT) for activity

• Storage conditions:

  • Temperature: -20 to -70°C for long-term storage

  • Avoid repeated freeze-thaw cycles

  • Use manual defrost freezers

• Sample preparation:

  • Cell/tissue lysis conditions must maintain enzyme integrity

  • Process samples rapidly to minimize degradation

  • Consider protease inhibitor cocktails (excluding thiol-reactive components)

• Assay conditions:

  • pH optimum should be determined empirically

  • Temperature stability (typically assayed at 37°C)

  • Substrate concentration relative to Km values

  • Incubation time within linear range of activity

• Controls:

  • Positive controls: recombinant PGPEP1 with verified activity

  • Negative controls: heat-inactivated enzyme

  • Background controls: substrate auto-hydrolysis rates

How does the evolutionary conservation of PGPEP1 impact translational research?

The high degree of sequence homology between human and mouse PGPEP1 (95% amino acid identity) has significant implications for translational research:

• Model system validity: Mouse models likely recapitulate the fundamental biological functions of PGPEP1, supporting their use in studying:

  • Physiological roles

  • Disease mechanisms

  • Therapeutic interventions

• Reagent cross-reactivity: Antibodies and other research tools often recognize both human and mouse PGPEP1, as demonstrated by antibodies detecting the protein in both species . This facilitates:

  • Cross-species validation

  • Translation between model systems

  • Reduction in required reagent development

• Functional conservation implications:

  • Substrate specificity likely maintained across species

  • Regulatory mechanisms potentially conserved

  • Disease-associated mechanisms may translate between models

• Therapeutic development considerations:

  • Drug screening platforms can use either human or mouse protein

  • Mouse studies likely predictive of human responses

  • Species differences (5% sequence divergence) may be exploited for selective targeting if needed

What contradictions exist in the current literature regarding PGPEP1 function?

The literature reveals several apparent contradictions regarding PGPEP1 that warrant further investigation:

These contradictions highlight important research gaps that present opportunities for significant contributions to the field.