XPNPEP1 Human

What is XPNPEP1 and what is its primary biochemical function?

XPNPEP1, also known as X-Prolyl Aminopeptidase 1, is a cytosolic metalloaminopeptidase that catalyzes the cleavage of N-terminal amino acids adjacent to proline residues. This enzyme plays a crucial role in the degradation and maturation of various bioactive peptides, including tachykinins, neuropeptides, and peptide hormones . As a member of the M24 family of metalloproteases, XPNPEP1 represents the soluble form of aminopeptidase P, in contrast to the GPI-anchored XPNPEP2 encoded by a separate gene . The enzyme has significant physiological importance due to its ability to hydrolyze substrates containing penultimate proline residues, such as bradykinin .

What are the common alternative names and identifiers for XPNPEP1?

Researchers should be aware of multiple nomenclature variants when searching literature:

This enzyme is classified as EC 3.4.11.9 . Using these alternative identifiers is essential for comprehensive literature searches and database mining.

What is the structural composition of human XPNPEP1 protein?

Human XPNPEP1 is a single, non-glycosylated polypeptide chain containing 623 amino acids with a molecular mass of approximately 73.4 kDa. Recombinant versions typically incorporate additional elements such as N-terminal or C-terminal His-tags for purification purposes. For instance, commercially available recombinant XPNPEP1 often contains 655 amino acids (positions 1-623 of the native sequence plus a 32 amino acid His-tag at the N-terminus) . The protein belongs to the peptidase M24B family, which shares structural features including metal-binding domains critical for enzymatic activity .

How can recombinant human XPNPEP1 be expressed and purified for research applications?

Recombinant human XPNPEP1 is commonly expressed in E. coli expression systems. The typical methodology involves:

• Cloning the human XPNPEP1 cDNA (positions 1-623) into a suitable expression vector containing an N-terminal or C-terminal His-tag

• Transforming the construct into an E. coli expression strain

• Inducing protein expression under optimized conditions

• Lysing cells and purifying the protein using proprietary chromatographic techniques, particularly nickel affinity chromatography targeting the His-tag

• Further purification may involve additional chromatographic steps to achieve >85% purity as determined by SDS-PAGE

The purified protein typically appears as a sterile filtered colorless solution, often formulated in phosphate-buffered saline (pH 7.4) containing 20% glycerol to enhance stability .

What assays can be used to measure XPNPEP1 enzymatic activity?

A validated fluorescence-based assay for measuring XPNPEP1 enzymatic activity utilizes the synthetic substrate Lys(ABZ)Pro-Pro-PNA. The detailed protocol includes:

• Prepare assay buffer (often a buffered solution containing required metal cofactors)

• Dilute purified XPNPEP1 to 0.5 ng/μL in assay buffer

• Prepare substrate solution (Lys(ABZ)Pro-Pro-PNA) to 100 μM in assay buffer

• In a F16 Black Maxisorp Plate, add 50 μL of enzyme solution (25 ng/well)

• Initiate the reaction by adding 50 μL of substrate solution

• Include substrate blanks (50 μL assay buffer + 50 μL substrate)

• Measure fluorescence at excitation/emission wavelengths of 320 nm and 410 nm in kinetic mode for 5 minutes

• Calculate specific activity based on the initial rate of substrate cleavage

This assay allows for quantitative assessment of enzymatic activity and can be used to evaluate potential inhibitors or study protein variants.

What are the optimal storage conditions for maintaining XPNPEP1 stability?

Maintaining XPNPEP1 stability is crucial for reliable experimental results. Recommended storage conditions include:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• Store frozen at -20°C for longer periods

• For long-term storage, add a carrier protein (0.1% HSA or BSA) to prevent protein loss through adsorption and maintain activity

• Avoid multiple freeze-thaw cycles as these can significantly reduce enzymatic activity

• Working solutions should be prepared fresh for each experiment

For recombinant proteins used in enzymatic assays, it's advisable to make single-use aliquots to prevent activity loss from repeated freezing and thawing.

How does XPNPEP1 deficiency affect neurological function?

XPNPEP1 deficiency has significant neurological consequences as demonstrated in knockout mouse models (Xpnpep1 -/-). These animals exhibit:

• Hippocampal neurodegeneration, particularly in the CA3 region

• Behavioral hyperactivity

• Impaired hippocampus-dependent learning and memory

• Markedly enhanced GluN1 and GluN2A expression

• Increased NMDAR activity

• Enhanced NMDAR-dependent long-term potentiation (LTP) of excitatory synaptic transmission

Mechanistically, the absence of aminopeptidase P1 results in massive urinary excretion of undigested peptides containing a penultimate proline residue. While the specific pathogenic peptides remain unidentified, the neurological phenotype appears to involve dysregulation of NMDAR homeostasis. Importantly, the NMDAR antagonist memantine reverses the exaggerated NMDAR activity and NMDAR-dependent LTP. A single administration of memantine can reverse hyperactivity in adult Xpnpep1-deficient mice, while chronic administration ameliorates hippocampal neurodegeneration, hyperactivity, and cognitive deficits .

This suggests that XPNPEP1 plays a critical role in maintaining synaptic function through modulation of NMDAR signaling, and pharmacological targeting of this pathway may represent a therapeutic approach for treating neurological complications related to inborn errors of metabolism affecting XPNPEP1.

What is known about XPNPEP1's role in cancer biology?

XPNPEP1 has emerged as a protein of interest in cancer research, with differential expression patterns observed in several malignancies:

• In clear cell renal cell carcinoma (ccRCC), particularly in patients with von Hippel-Lindau disease, XPNPEP1 shows increased abundance compared to adjacent non-malignant kidney tissue. This is notable because it occurs concurrently with decreased expression of the related protease XPNPEP2 .

• Functional studies using small-hairpin RNA-mediated XPNPEP1 silencing in 786-O ccRCC cells (which harbor a mutated VHL gene) revealed that XPNPEP1 expression dampens cellular proliferation and migration. This suggests that XPNPEP1 may function as an anti-target in ccRCC, meaning that inhibition might actually promote cancer progression .

• Outside of renal cancer, XPNPEP1 has been associated with unfavorable outcomes in acute myeloid leukemia, though reports of XPNPEP1 in cancer biology remain limited .

These findings indicate complex and potentially context-dependent roles for XPNPEP1 in different cancer types, warranting further investigation into its mechanistic contributions to tumor biology and its potential as a biomarker or therapeutic target.

How can CRISPR technologies be applied to study XPNPEP1 function?

CRISPR-Cas9 technology offers powerful approaches for investigating XPNPEP1 function:

• Gene Knockout Studies: Complete ablation of XPNPEP1 expression allows assessment of phenotypic consequences in cellular models. According to BioGRID ORCS database, XPNPEP1 has been included in 27 CRISPR screens out of 1368 screens cataloged . These screens provide valuable information about cellular contexts where XPNPEP1 may be essential or dispensable.

• Domain-Specific Mutations: CRISPR-mediated introduction of point mutations can target catalytic domains or protein interaction sites to distinguish enzymatic from non-enzymatic functions.

• Endogenous Tagging: Insertion of fluorescent or affinity tags at the endogenous locus enables visualization of native expression patterns and identification of protein complexes without overexpression artifacts.

• Inducible Systems: Combining CRISPR with inducible promoters allows temporal control of XPNPEP1 disruption, facilitating study of acute versus chronic loss.

• Paralog Comparison: Simultaneous or sequential targeting of XPNPEP1 and XPNPEP2 can reveal redundant versus unique functions of these related aminopeptidases.

When designing CRISPR studies, researchers should consider potential off-target effects and validate editing efficiency through sequencing and protein detection methods.

What methodological approaches can distinguish between XPNPEP1 and XPNPEP2 functions?

Despite similarities in catalytic activity, XPNPEP1 (cytosolic) and XPNPEP2 (membrane-anchored) have distinct subcellular localizations and potentially different physiological roles. Methodological approaches to distinguish their functions include:

• Subcellular Fractionation: Separate cytosolic (XPNPEP1-containing) and membrane (XPNPEP2-containing) fractions before enzymatic assays to determine compartment-specific activity.

• Selective Inhibition: While both enzymes are inhibited by apstatin , development of isoform-selective inhibitors through structure-based design could enable specific targeting. Differences in protein structure might be exploited for selective inhibition.

• Cellular Models: Compare phenotypes between XPNPEP1-knockout, XPNPEP2-knockout, and double-knockout models to identify unique and overlapping functions.

• Substrate Profiling: Employ peptide libraries or targeted metabolomics to identify differential substrate preferences between the two enzymes in physiological contexts.

• Expression Studies: Analyze tissue-specific expression patterns and regulation mechanisms, as differential expression may indicate tissue-specific functions.

These approaches can help elucidate the distinct physiological roles of these aminopeptidases and their potential as therapeutic targets in different disease contexts.

How do post-translational modifications regulate XPNPEP1 activity?

While specific information about post-translational modifications (PTMs) of XPNPEP1 is limited in the provided search results, general methodological approaches to investigate PTMs include:

• Mass Spectrometry: Employ high-resolution MS to identify phosphorylation, acetylation, ubiquitination, or other modifications. FFPE proteomics approaches have been successfully used for analyzing proteomic profiles including XPNPEP1 in clinical samples .

• Site-Directed Mutagenesis: Mutate potential modification sites to mimic or prevent modifications (e.g., phosphomimetic substitutions) and assess functional consequences.

• Pharmacological Intervention: Use kinase inhibitors, deacetylase inhibitors, or proteasome inhibitors to alter modification status and monitor effects on enzymatic activity.

• Modification-Specific Antibodies: Develop or employ antibodies that recognize specific PTMs of XPNPEP1 for immunoblotting or immunoprecipitation studies.

• In Vitro Modification: Perform in vitro enzymatic reactions to determine if specific modifications alter substrate recognition or catalytic efficiency.

Understanding the regulatory mechanisms controlling XPNPEP1 activity through PTMs could reveal new therapeutic opportunities and explain context-dependent functions of this enzyme.