ACP5 Human, His

What is the molecular identity of ACP5 and its nomenclature in research literature?

ACP5 is known by several names in the scientific literature, including Tartrate-Resistant Acid Phosphatase (TRACP or TRAP) and Purple Acid Phosphatase (PAP). The human ACP5 protein consists of a signal peptide (amino acids 1-21) and a mature chain (amino acids 22-325). When working with recombinant human ACP5, researchers should note that commercial preparations (such as R&D Systems) typically contain amino acids 22-320, omitting the last 5 residues (RRARP) .

ACP5 is a secreted phosphatase enzyme that belongs to the acid phosphatase family and is distinguished by its resistance to inhibition by tartrate, a characteristic that differentiates it from other acid phosphatases. This feature is particularly important when designing experimental protocols for selective detection and activity measurement of ACP5 among other phosphatases.

Which cell types predominantly express ACP5 and what are the distinct circulating forms?

ACP5 is expressed at high levels by three primary cell types: osteoclasts, macrophages, and dendritic cells . The differential expression in these cell populations contributes to the protein's diverse biological functions. In human circulation, ACP5 exists in two distinct forms that serve as biomarkers for different physiological processes:

• Form 5a: Derived from macrophages and dendritic cells, serving as a marker of inflammatory conditions

• Form 5b: Derived from osteoclasts, functioning as a marker of bone resorption

Researchers investigating ACP5 should carefully consider which form they are targeting, as the biological significance of each differs substantially depending on the research context.

What are the validated experimental methods for detecting ACP5 protein expression in research samples?

Multiple validated techniques exist for detecting ACP5 protein expression in research samples:

• Western Blotting:

  • Detects ACP5 at approximately 36 kDa under reducing conditions

  • Recommended antibodies include Sheep Anti-Human TRACP/PAP/ACP5 Antigen Affinity-purified Polyclonal Antibody (e.g., R&D Systems catalog #AF3948)

  • PVDF membranes are preferred with HRP-conjugated secondary antibodies

• Immunohistochemistry (IHC):

  • Particularly useful for tissue localization studies

  • In pancreatic cancer studies, ACP5 staining appears as light yellow and tan in para-cancerous tissues and shifts to tan and brown in cancerous tissues

  • Quantification can be performed using image analysis software such as ImageJ to measure the percentage of DAB-colored regions

• Enzyme Activity Assays:

  • Utilizing p-Nitrophenyl phosphate as substrate

  • Performed in acidic conditions (pH 5.0) to optimize enzyme activity

  • Specific activity calculated using spectrophotometric measurements at 410 nm

How is ACP5 expression regulated at the epigenetic level?

Research demonstrates that ACP5 expression is significantly influenced by epigenetic mechanisms, particularly DNA methylation. Studies in pancreatic cancer have revealed an inverse correlation between ACP5 expression and promoter methylation levels . The UALCAN analysis found consistently lower levels of methylated ACP5 in tumor tissues compared to normal tissues, suggesting that hypomethylation of the ACP5 promoter is a key mechanism driving its increased expression in cancer .

This epigenetic regulation varies across different clinicopathologic subgroups. For instance, reduced methylation of ACP5 was observed across diverse patient characteristics, including:

• Patients with lymph node metastasis

• Female patients (showing significantly lower levels compared to normal controls)

• Different age groups, smoking status categories, tumor grades, cancer stages, and P53 mutation status

These findings suggest that researchers investigating ACP5 expression should consider epigenetic regulation as a critical factor in experimental design and data interpretation.

What is the pattern of ACP5 expression in cancer versus normal tissues?

ACP5 exhibits consistent upregulation across multiple cancer types compared to corresponding normal tissues:

• In lung adenocarcinoma (LUAD):

  • Increased expression in 57.97% (40/69) of tumor samples compared to adjacent non-cancerous tissues

  • Higher expression in NCI-H1975, H1299, and A549 cancer cell lines compared to normal Beas-2b cells

• In pancreatic cancer (PC):

  • Significantly elevated expression in tumor tissues versus para-cancerous tissues

  • Higher expression in cancer cell lines (PANC-1, BXPC3, CFPAC-1, ASPC-1) compared to normal pancreatic epithelial cells (hTERT-HPNE)

  • Upregulation confirmed through multiple analytical approaches including GTEx and TCGA data analysis, TNM plot, and GEPIA

This consistent pattern of upregulation across different malignancies suggests a fundamental role for ACP5 in cancer biology that transcends specific tissue contexts.

How does ACP5 expression correlate with clinicopathological features in cancer patients?

ACP5 expression demonstrates significant correlations with several clinicopathological features, suggesting its potential as a biomarker for disease progression and patient stratification. Key correlations include:

In lung adenocarcinoma:

• Patient age (p = 0.044): Higher expression in patients >58 years (36.23%) compared to ≤58 years (21.74%)

• Lymph node metastasis (p = 0.0385)

In pancreatic cancer:

• Tumor location: High ACP5 expression tumors were predominantly found at the head of the pancreas

• Disease stage: Higher ACP5 expression correlated with advanced T stage and pathologic stage, indicating more extensive malignancy

These correlations provide valuable insights for researchers studying the prognostic significance of ACP5 in different cancer types.

How can researchers effectively measure ACP5 enzymatic activity in experimental settings?

A standardized protocol for measuring ACP5 enzymatic activity utilizes p-Nitrophenyl phosphate as a substrate with spectrophotometric detection:

Materials Required:

• Assay Buffer: 50 mM NaOAc, pH 5.0

• Recombinant Human TRACP/PAP/ACP5 (rhACP5)

• Substrate: p-Nitrophenyl phosphate

• 96-well Clear Plate

• Plate Reader (capable of reading at 410 nm)

• NaOH, 0.2 M in deionized water

Procedure:

• Dilute rhACP5 to 0.1 μg/mL in Assay Buffer

• Dilute Substrate to 2 mM in Assay Buffer

• In a plate, combine 50 μL of rhACP5 and 50 μL of 2 mM Substrate; include a Substrate Blank (50 μL Assay Buffer + 50 μL Substrate)

• Incubate at room temperature for 10 minutes in the dark

• Add 100 μL of 0.2 M NaOH to stop the reaction and develop color

• Read absorbance at 410 nm

Activity Calculation:
Specific Activity (pmol/min/μg) = [Adjusted Abs (OD) × Conversion Factor (pmol/OD)] / [Incubation time (min) × amount of enzyme (μg)]

This standardized method allows for reproducible quantification of ACP5 activity across different experimental conditions and laboratory settings.

What experimental approaches are recommended for investigating ACP5's role in cell proliferation and migration?

Based on published research, several validated experimental approaches can be employed to investigate ACP5's role in cell proliferation and migration:

• Gene Manipulation Techniques:

  • RNA interference (siRNA treatment) to efficiently knock down ACP5 expression

  • Plasmid-mediated overexpression of ACP5

• Proliferation Assays:

  • Cell Counting Kit-8 (CCK8) assay to measure cell proliferation rates

  • Clonogenic assays to assess independent cell viability and colony-forming capacity

• Migration and Invasion Assays:

  • Wound healing assays to evaluate cellular motility over time

  • Transwell migration assays to quantify directional cell movement

  • Matrigel invasion assays to assess invasive capacity through extracellular matrix components

• Apoptosis Analysis:

  • Flow cytometry to measure apoptosis rates under various conditions (e.g., H₂O₂-induced stress)

  • Combination with ACP5 knockdown or overexpression to determine protective effects

• In Vivo Models:

  • Mouse models with manipulated ACP5 expression to evaluate tumor growth and metastasis

  • Assessment of intrapulmonary metastasis following ACP5 overexpression

These complementary approaches provide a comprehensive evaluation of ACP5's functional impact on cancer cell behavior.

What considerations are important when working with recombinant human ACP5 in experimental systems?

When utilizing recombinant human ACP5 (rhACP5) in experimental systems, researchers should consider several critical factors:

• Protein Composition:

  • Commercial rhACP5 typically comprises amino acids 22-320, lacking the signal peptide (aa 1-21) and the C-terminal 5 residues (RRARP)

  • This composition difference from the native protein may affect certain interaction studies

• Storage and Stability:

  • Use manual defrost freezers to avoid freeze-thaw damage

  • Optimal storage conditions: -20 to -70°C for 12 months from receipt (as supplied)

  • After reconstitution: 1 month at 2-8°C or 6 months at -20 to -70°C under sterile conditions

• Enzymatic Activity Considerations:

  • Optimal activity at acidic pH (5.0)

  • Activity varies with substrate concentration; recommended working concentration: 0.005 μg rhACP5 with 0.5 mM substrate

  • Include appropriate controls for non-specific phosphatase activity

• Potential Contaminants:

  • Evaluate purification method and source to avoid activity from contaminating phosphatases

  • Verify tartrate resistance to confirm specific ACP5 activity

• Post-translational Modifications:

  • Consider the expression system used for producing rhACP5 as this may affect glycosylation and other modifications

  • These modifications could impact protein activity, stability, and biological functions

What is the mechanistic pathway through which ACP5 regulates p53 function?

Research has elucidated a detailed mechanistic pathway through which ACP5 regulates p53 function, primarily affecting its stability rather than transcription:

• ACP5 regulates p53 phosphorylation specifically at Serine 392, a critical post-translational modification site

• This phosphorylation change enhances p53 ubiquitination, targeting it for proteasomal degradation

• Evidence for the ubiquitin-proteasome pathway involvement:

  • Treatment with MG132 (a proteasome inhibitor) reverses ACP5-induced downregulation of p53

  • Immunoprecipitation assays demonstrate significantly enhanced p53 ubiquitination in ACP5-transfected A549 cells

  • Quantitative real-time PCR confirms that ACP5 affects p53 protein stability rather than transcriptional regulation

• Downstream consequences:

  • Reduced p53 levels intensify SMAD3 transcription

  • Enhanced SMAD3 expression promotes epithelial-mesenchymal transition (EMT)

  • This pathway ultimately contributes to increased cancer cell invasion and metastasis

This mechanistic understanding provides crucial insights for researchers exploring potential therapeutic interventions targeting the ACP5-p53 axis.

How does ACP5 influence the epithelial-mesenchymal transition (EMT) process in cancer cells?

ACP5 exerts multifaceted effects on the epithelial-mesenchymal transition (EMT) process in cancer cells:

• Direct experimental evidence:

  • ACP5 overexpression significantly enhances EMT in lung cancer cell lines (A549 and NCI-H1975)

  • Knockdown of ACP5 reverses these EMT-promoting effects

• Signaling pathway interactions:

  • ACP5 operates downstream of forkhead box M1 (Foxm1), which is required for lung fibrosis and EMT in tumor metastasis

  • The ACP5-p53-SMAD3 axis represents a key mechanism:

    • ACP5 promotes p53 degradation

    • Reduced p53 levels enhance SMAD3 transcription

    • Increased SMAD3 acts as a potent EMT inducer

• Clinical correlations:

  • In lung adenocarcinoma, ACP5 expression correlates with lymph node metastasis (p = 0.0385)

  • This suggests that ACP5-induced EMT contributes to metastatic spread

• In vivo validation:

  • Enhanced ACP5 expression promotes hyperplasia and intrapulmonary metastasis in mouse models

  • This confirms the functional relevance of ACP5-mediated EMT in cancer progression

Understanding these mechanisms provides researchers with potential intervention points for targeting the EMT process in cancer cells.

What is the relationship between ACP5 and the tumor immune microenvironment?

Research has begun to uncover important relationships between ACP5 and the tumor immune microenvironment, particularly in pancreatic cancer:

• Immune cell infiltration:

  • ACP5 expression positively correlates with immune cell infiltration in pancreatic cancer

  • This correlation is particularly strong with regulatory T cells (Tregs), which typically suppress anti-tumor immune responses

• Analytical approaches:

  • This relationship has been established using sophisticated computational methods:

    • Tumor Immune Estimation Resource (TIMER) analysis

    • R programming-based correlation analyses

• Cellular sources of ACP5 in the immune context:

  • ACP5 is highly expressed by macrophages and dendritic cells, which are key components of the tumor immune microenvironment

  • The 5a form of circulating ACP5 (derived from these immune cells) serves as a marker of inflammatory conditions

• Potential implications:

  • ACP5 may contribute to immune evasion mechanisms in cancer

  • The correlation with Tregs suggests a potential immunosuppressive role

  • This relationship could impact the efficacy of immunotherapeutic approaches

These findings suggest that researchers investigating cancer immunology should consider ACP5 as a potential modulator of the tumor immune microenvironment.

How does ACP5 expression impact cancer patient prognosis across different tumor types?

ACP5 expression has been linked to prognostic outcomes across multiple cancer types:

• Lung Adenocarcinoma (LUAD):

  • Increased ACP5 levels associate with lymph node metastasis (p = 0.0385)

  • Higher expression correlates with older patient age (p = 0.044)

• Pancreatic Cancer (PC):

  • ACP5 expression increases with advanced disease stage

  • Higher expression correlates with more extensive malignancy in terms of T stage and pathologic stage

  • Expression patterns vary by tumor location, with high expression tumors often located at the head of the pancreas

• Other Malignancies:

  • ACP5 serves as a useful serum marker for extensive bone metastasis in melanoma

  • Expression is significantly upregulated in breast cancer, hepatocellular carcinoma, and ovarian cancer

  • Elevated ACP5 generally indicates poor prognosis across these malignancies

These consistent prognostic correlations across diverse tumor types suggest a fundamental role for ACP5 in cancer progression, making it a potentially valuable biomarker for patient stratification and treatment planning.

What experimental evidence supports ACP5 as a potential therapeutic target in cancer?

Multiple lines of experimental evidence support ACP5 as a promising therapeutic target in cancer:

These collective findings provide a strong rationale for developing therapeutic strategies targeting ACP5 in cancer.

What methodological challenges exist in targeting ACP5 for cancer therapy?

Several methodological challenges must be addressed when considering ACP5 as a therapeutic target:

• Specificity considerations:

  • ACP5 belongs to the acid phosphatase family with structural similarities to other phosphatases

  • Developing inhibitors with sufficient specificity for ACP5 without affecting related phosphatases remains challenging

  • Careful enzyme kinetic studies and selectivity profiling are necessary during inhibitor development

• Expression in normal tissues:

  • ACP5 is expressed in normal osteoclasts, macrophages, and dendritic cells

  • Therapeutic targeting must minimize effects on these normal cell populations

  • Strategies might include cancer-specific delivery systems or exploiting differential expression levels

• Functional redundancy:

  • Other phosphatases may compensate for ACP5 inhibition

  • Combination approaches targeting multiple nodes in the pathway may be necessary

  • Comprehensive phosphatase profiling in target tissues would inform such strategies

• Biomarker development:

  • Patient selection for ACP5-targeted therapies requires reliable biomarkers

  • Standardization of ACP5 detection methods across clinical laboratories

  • Determination of clinically relevant expression thresholds for patient stratification

• Delivery to target tissues:

  • Ensuring sufficient drug concentration in tumor tissue

  • Consideration of the blood-brain barrier for potential brain metastases

  • Development of innovative delivery systems for phosphatase inhibitors

Addressing these challenges requires multidisciplinary approaches combining structural biology, medicinal chemistry, pharmacology, and clinical oncology expertise.