CPPED1 Human

What is the structural classification of human CPPED1?

CPPED1 contains a calcineurin-like phosphoesterase domain, but sequence homology analyses reveal it does not belong to the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) families as previously thought. Instead, CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily . Sequence comparisons show CPPED1's closest homologs are 3',5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase from Synechococcus sp (47% similarity) and purple acid phosphatase 22 from Arabidopsis thaliana (42% similarity) . Importantly, CPPED1 lacks the two active-site loops characteristic of PPP and PPM family members, further supporting its classification in the PDE subfamily.

What are the known enzymatic functions of CPPED1?

CPPED1 functions as a serine/threonine protein phosphatase that dephosphorylates AKT1 at Ser473 in the PI3K-AKT signaling pathway . Its phosphatase activity is cation-dependent, showing higher enzymatic activity in the presence of Mn²⁺ compared to Ca²⁺ or no cations . Research has also demonstrated that CPPED1 can dephosphorylate specific serine residues in PAK4 (p21 [RAC1] activated kinase 4), while having no effect on the phosphorylation levels of PIK3R2 (phosphoinositide-3-kinase regulatory subunit 2) . These enzymatic activities suggest CPPED1 regulates the PI3K-AKT pathway at multiple levels by targeting different components of the signaling cascade.

How is recombinant CPPED1 purified for functional studies?

The purification of recombinant CPPED1 involves several key steps:

• Cloning of CPPED1 into a constitutively active expression plasmid (e.g., pSFOXB20)

• Expression of the recombinant protein with an N-terminal His-V5 dual tag in E. coli BL21 (DE3) cells

• Purification through a combination of:

  • Affinity chromatography

  • Hydrophobic interaction chromatography

  • Size-exclusion chromatography

• Verification of protein purity by SDS-gel electrophoresis

After purification, proper folding is confirmed using circular dichroism (CD) spectroscopy, which reveals that human recombinant CPPED1 comprises both α-helical and β-sheet structures . Static light scattering (SLS) measurements confirm the protein exists as a homogenous, monomeric sample in solution. Enzymatic activity is verified through in vitro phosphatase assays with appropriate cations, particularly Mn²⁺ .

What protein-protein interactions have been identified for CPPED1 and what methods were used to discover them?

A comprehensive human proteome microarray (HuProt™ v3.1) was used to identify 36 proteins that interact with CPPED1 in vitro . This microarray covers approximately 75% of the annotated human protein-coding genome. The identified interacting proteins include:

The interactions with PAK4 and PIK3R2 were further confirmed using coimmunoprecipitation (CoIP) and bimolecular fluorescence complementation (BiFC) methods, verifying these interactions occur in vivo . Mass spectrometry analysis was subsequently used to characterize the effect of CPPED1 on the phosphorylation states of these interacting proteins, revealing that CPPED1 dephosphorylates specific serine residues in PAK4 while having no effect on PIK3R2 phosphorylation .

How does CPPED1 regulate the PI3K-AKT signaling pathway at the molecular level?

CPPED1 regulates the PI3K-AKT pathway through multiple mechanisms:

• Direct dephosphorylation of AKT1: CPPED1 dephosphorylates AKT1 at Ser473, inhibiting its activity . This dephosphorylation prevents cancer progression in bladder cancer models.

• Interaction with pathway components: Proteome microarray and subsequent confirmatory experiments revealed that CPPED1 interacts with key regulators of the PI3K-AKT pathway, including:

  • GRB2 (growth factor receptor bound protein 2)

  • PAK4 (p21 [RAC1] activated kinase 4)

  • PIK3R2 (phosphoinositide-3-kinase regulatory subunit 2)

• Dephosphorylation of PAK4: CPPED1 dephosphorylates specific serine residues in PAK4, which is known to bind to PIK3R1 and activate the PI3K pathway .

• Transcriptional regulation: Silencing of CPPED1 in HTR8/SVneo trophoblast cells leads to enhanced expression of negative regulatory genes of the PI3K pathway, such as PIK3IP1 (phosphoinositide-3-kinase interacting protein 1) and PIK3CG (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma) .

In functional terms, higher CPPED1 levels inhibit the PI3K-AKT pathway, which may play a role in maintaining pregnancy . Conversely, decreased CPPED1 expression, as observed during labor, may alter this regulation, though the specific consequences require further investigation.

What experimental approaches are used to study CPPED1's role in glucose metabolism and cancer?

Research into CPPED1's role in metabolism and cancer involves several sophisticated experimental approaches:

• Expression analysis in tissue samples:

  • Measurement of CPPED1 expression levels in normal vs. cancer tissues (particularly bladder cancer)

  • Correlation of expression levels with disease progression and tumor size

• Glucose uptake assays:

  • CPPED1 knockdown experiments using siRNA

  • Measurement of glucose uptake in control vs. knockdown cells

  • Assessment of the effects of PI3K-specific inhibitors (e.g., wortmannin) on glucose uptake in CPPED1 knockdown cells

• Signaling pathway analysis:

  • Western blotting to detect phosphorylation states of AKT and other PI3K pathway components

  • Transcriptomic analyses to identify genes regulated by CPPED1

  • Treatment with pathway inhibitors to establish causality between CPPED1 activity and downstream effects

Research has shown that increased glucose uptake is associated with decreased CPPED1 expression, and that treatment with wortmannin decreases glucose uptake in CPPED1 knockdown cells, suggesting CPPED1 mediates glucose metabolism via the PI3K-AKT signaling pathway .

What is known about CPPED1's role in pregnancy and labor?

CPPED1 has been identified as a potential regulator of pregnancy maintenance and labor initiation:

• Expression patterns: CPPED1 levels are down-regulated in the human placenta during spontaneous term birth , suggesting a potential role in the mechanisms of labor onset.

• Cellular studies: In HTR8/SVneo trophoblast cells:

  • Silencing of CPPED1 expression leads to enhanced expression of negative regulatory genes of the PI3K pathway

  • CPPED1 mediates the effect of progesterone on gene expression

• Mechanistic hypothesis: Higher CPPED1 levels may inhibit the PI3K-AKT pathway, which appears to be important for maintaining pregnancy . The decrease in CPPED1 expression during labor may alter this inhibition, potentially contributing to labor onset.

• Progesterone-mediated effects: Treatment of HTR8/SVneo cells with progesterone (P4) alters the expression of 98 genes (46 down-regulated, 52 up-regulated) . Silencing of CPPED1 expression removes this effect of progesterone, indicating CPPED1 mediates progesterone's transcriptional effects.

The exact mechanisms by which CPPED1 influences labor onset and pregnancy maintenance require further investigation, but the connection to progesterone signaling and the PI3K-AKT pathway suggests important regulatory functions.

How can researchers confirm protein-protein interactions involving CPPED1?

Multiple complementary approaches are recommended for confirming protein-protein interactions with CPPED1:

• Large-scale screening via protein microarray:

  • Use of human proteome microarrays (e.g., HuProt™) for initial identification

  • Incubation of purified recombinant CPPED1 with the microarray

  • Detection of interactions using fluorescently labeled antibodies against CPPED1 tags

• Coimmunoprecipitation (CoIP):

  • Expression of tagged versions of CPPED1 and potential interacting proteins

  • Immunoprecipitation using antibodies against the tag

  • Western blot analysis to detect coprecipitated proteins

  • Bidirectional confirmation (precipitate with anti-CPPED1 and detect interactor, and vice versa)

• Bimolecular fluorescence complementation (BiFC):

  • Fusion of CPPED1 and interactor with complementary fragments of a fluorescent protein

  • Co-expression in mammalian cells

  • Microscopic detection of reconstituted fluorescence indicating protein interaction in vivo

• Mass spectrometry analysis:

  • In vitro incubation of CPPED1 with purified interacting proteins

  • Analysis of phosphorylation state changes to confirm functional interactions

  • Detection of specific modified residues to understand mechanistic details

Each method has strengths and limitations, and combining multiple approaches provides more robust evidence for protein-protein interactions.

What functional assays can be used to evaluate CPPED1's phosphatase activity?

Evaluating CPPED1's phosphatase activity requires specific functional assays:

• In vitro phosphatase assays:

  • Incubation of purified recombinant CPPED1 with phosphorylated substrates (e.g., AKT1, PAK4)

  • Inclusion of appropriate cations (Mn²⁺, Ca²⁺) to test cation dependency

  • Measurement of released phosphate or detection of substrate dephosphorylation via Western blotting

• Mass spectrometry-based approaches:

  • Incubation of CPPED1 with potential substrates in vitro

  • Digestion of proteins and analysis of phosphopeptides

  • Identification of specific dephosphorylated residues

  • Quantitative comparison of phosphorylation levels with and without CPPED1

• Cellular phosphorylation assays:

  • Overexpression or knockdown of CPPED1 in relevant cell lines

  • Stimulation of signaling pathways (e.g., with growth factors)

  • Western blot analysis of phosphorylation states of potential substrates

  • Inclusion of phosphatase inhibitors as controls

• Enzyme kinetics studies:

  • Determination of reaction rates with varying substrate concentrations

  • Calculation of kinetic parameters (Km, Vmax)

  • Analysis of inhibition patterns with various inhibitors

  • Evaluation of the effects of different metal ions on enzymatic activity

These assays should be performed with appropriate controls, including phosphatase-dead CPPED1 mutants and known phosphatase inhibitors, to ensure specificity and reliability of results.

How can researchers investigate the transcriptional effects of CPPED1 in cell-based models?

Investigating CPPED1's transcriptional effects requires systematic approaches:

• RNA interference experiments:

  • Transfection of cells with siRNA targeting CPPED1

  • Verification of knockdown efficiency at mRNA (qRT-PCR) and protein (Western blot) levels

  • Typical knockdown efficiency should be at least 60-70% for meaningful results

• Overexpression studies:

  • Transfection with CPPED1 expression constructs

  • Creation of stable cell lines with inducible CPPED1 expression

  • Verification of expression using qRT-PCR and Western blotting

• Transcriptomic analysis:

  • RNA sequencing of control vs. CPPED1-modulated cells

  • Differential expression analysis to identify regulated genes

  • Pathway enrichment analysis to identify affected biological processes

  • Validation of key findings by qRT-PCR

• Hormone response studies:

  • Treatment of cells with relevant hormones (e.g., progesterone)

  • Comparison of hormone effects in control vs. CPPED1-silenced cells

  • Identification of CPPED1-dependent hormone-responsive genes

• Promoter analysis:

  • Reporter assays using promoter regions of CPPED1-regulated genes

  • Chromatin immunoprecipitation (ChIP) to identify transcription factors involved

  • Analysis of signaling pathway activation (e.g., PI3K-AKT) in relation to transcriptional changes

These approaches help establish causal relationships between CPPED1 levels and downstream transcriptional effects, providing insight into its biological functions.

What are potential therapeutic implications of targeting CPPED1 in cancer?

CPPED1 shows promising therapeutic potential in cancer contexts:

• Tumor suppressor activity:

  • CPPED1 dephosphorylates AKT1 at Ser473, preventing cancer progression in bladder cancer

  • CPPED1 expression levels are down-regulated in non-invasive bladder cancer tissue

  • Overexpression of CPPED1 is associated with regression in tumor size

• Therapeutic strategies:

  • Gene therapy approaches to restore CPPED1 expression in cancers with low CPPED1 levels

  • Small molecule activators of CPPED1 phosphatase activity

  • Targeted delivery of CPPED1 protein to tumor cells

• Combination therapy opportunities:

  • CPPED1-based therapies could potentially enhance the efficacy of existing PI3K-AKT pathway inhibitors

  • Synergistic effects might be achieved with mTOR inhibitors or other agents targeting this pathway

• Biomarker potential:

  • CPPED1 expression levels could serve as prognostic or predictive biomarkers in certain cancers

  • Phosphorylation status of CPPED1 substrates might indicate pathway activity

• Potential challenges:

  • Tissue-specific effects of CPPED1 must be considered

  • Systemic activation of CPPED1 could potentially affect glucose metabolism and other physiological processes

  • Optimal methods for targeting CPPED1 specifically in tumor cells need development

Research into CPPED1's role in different cancer types and detailed mechanistic studies are needed to fully exploit its therapeutic potential.

How might CPPED1 contribute to metabolic disorders, and what research approaches could elucidate this relationship?

CPPED1's role in glucose metabolism suggests potential involvement in metabolic disorders:

• Current knowledge:

  • CPPED1 regulates glucose uptake in adipose tissue

  • Decreased CPPED1 expression is associated with increased glucose uptake

  • CPPED1 mediates glucose metabolism via the PI3K-AKT signaling pathway

• Research approaches to investigate metabolic functions:

  • Animal models:

    • Generation of tissue-specific CPPED1 knockout or overexpression mice

    • Phenotypic characterization focusing on glucose homeostasis and insulin sensitivity

    • Metabolic challenges (high-fat diet, glucose tolerance tests)

  • Human studies:

    • Genetic association studies of CPPED1 variants with metabolic phenotypes

    • Analysis of CPPED1 expression in metabolic tissues from patients with diabetes or obesity

    • Correlation of expression with clinical parameters

  • Cellular metabolism studies:

    • Glucose uptake assays in various cell types with modulated CPPED1 levels

    • Insulin signaling analysis with focus on PI3K-AKT pathway components

    • Lipolysis and lipogenesis measurements in adipocytes

• Potential relevance to metabolic disorders:

  • Type 2 diabetes: CPPED1 modulation could affect insulin sensitivity through its effect on the PI3K-AKT pathway

  • Obesity: Altered adipose tissue glucose metabolism might influence fat storage

  • Metabolic syndrome: Multiple aspects could be influenced through CPPED1's regulatory roles

These research directions could establish CPPED1 as a potential therapeutic target or biomarker for metabolic disorders, particularly those involving insulin resistance or glucose homeostasis.

What are the current challenges and limitations in CPPED1 research?

Despite progress in understanding CPPED1, several challenges and limitations exist:

• Technical challenges:

  • Obtaining highly purified, active recombinant CPPED1 requires multiple chromatography steps

  • Measuring phosphatase activity requires careful consideration of metal ion requirements

  • Identifying specific substrates among numerous phosphorylated proteins in cells

• Knowledge gaps:

  • The complete spectrum of CPPED1 substrates remains unidentified

  • Tissue-specific functions and expression patterns are incompletely characterized

  • Regulatory mechanisms controlling CPPED1 expression and activity are poorly understood

  • The three-dimensional structure of CPPED1 has not been reported

• Methodological limitations:

  • Lack of highly specific CPPED1 inhibitors or activators

  • Challenges in distinguishing CPPED1's effects from other phosphatases targeting similar substrates

  • Limited availability of phospho-specific antibodies for all potential CPPED1 substrates

• Translational barriers:

  • Incomplete understanding of how CPPED1 dysregulation contributes to human diseases

  • Limited knowledge of CPPED1 polymorphisms and their functional consequences

  • Need for better models to study CPPED1's role in pregnancy and labor

• Future research needs:

  • Development of conditional knockout models

  • Creation of specific pharmacological modulators of CPPED1 activity

  • High-resolution structural studies to facilitate drug design

  • Systems biology approaches to place CPPED1 in broader signaling networks

Addressing these challenges will require interdisciplinary approaches combining structural biology, biochemistry, cell biology, and translational research.

How does CPPED1 interact with the progesterone signaling pathway in pregnancy-related tissues?

The interaction between CPPED1 and progesterone signaling represents an emerging research area:

• Current evidence:

  • CPPED1 levels are down-regulated in the human placenta during spontaneous term birth

  • In HTR8/SVneo trophoblast cells, CPPED1 mediates the effect of progesterone on gene expression

  • Treatment with progesterone alters the expression of 98 genes, and this effect is removed by silencing CPPED1

• Research approaches to explore this interaction:

  • Receptor interaction studies:

    • Investigation of potential physical interactions between CPPED1 and progesterone receptor isoforms

    • Characterization of how progesterone affects CPPED1 phosphatase activity and substrate specificity

    • Analysis of whether CPPED1 directly affects progesterone receptor phosphorylation

  • Transcriptional regulation:

    • ChIP-seq studies to identify genomic binding sites of progesterone receptor in the presence/absence of CPPED1

    • Analysis of whether CPPED1 affects recruitment of transcriptional coregulators

    • Identification of progesterone-responsive genes dependent on CPPED1

  • Signaling crosstalk:

    • Investigation of how PI3K-AKT signaling interacts with progesterone receptor signaling

    • Determination of whether CPPED1 serves as an integration point between these pathways

    • Assessment of CPPED1's role in non-genomic progesterone effects

• Clinical relevance:

  • Understanding CPPED1's role in progesterone signaling could provide insights into preterm labor mechanisms

  • Potential for identifying novel therapeutic targets for pregnancy complications

  • Possible relevance to progesterone-dependent conditions beyond pregnancy

This research direction could significantly enhance our understanding of labor onset and maintenance of pregnancy, with potential implications for managing preterm birth risk.

What is the potential role of CPPED1 in inflammatory and immune responses?

While not extensively studied, several indicators suggest CPPED1 may play a role in immune and inflammatory processes:

• Pathway connections:

  • CPPED1 regulates the PI3K-AKT pathway, which is involved in both innate and adaptive immunity

  • CPPED1 interacts with immune-related proteins identified in the proteome microarray, including:

    • CXCL16 (C-X-C motif chemokine ligand 16)

    • ISG20 (Interferon-stimulated exonuclease gene 20)

    • IRF2BP2 (Interferon regulatory factor 2 binding protein 2)

• Research approaches to explore immune functions:

  • Immune cell studies:

    • Analysis of CPPED1 expression in different immune cell populations

    • Functional studies in macrophages, dendritic cells, and lymphocytes with modified CPPED1 levels

    • Assessment of cytokine production and inflammatory responses

  • Signaling analysis:

    • Investigation of how CPPED1 affects immune receptor signaling pathways

    • Analysis of potential roles in interferon signaling, given its interaction with interferon-related proteins

    • Examination of effects on NF-κB activation and other inflammatory transcription factors

  • Disease models:

    • Evaluation of CPPED1 expression and function in inflammatory disease models

    • Assessment of immune cell function in tissue-specific CPPED1 knockout models

    • Testing whether CPPED1 modulation affects disease progression or resolution

• Potential significance:

  • CPPED1 could represent a novel regulatory component in immune homeostasis

  • Its phosphatase activity might dampen excessive immune activation

  • Therapeutic targeting could potentially modulate inflammatory conditions

This unexplored aspect of CPPED1 biology warrants investigation and could reveal new roles beyond metabolism and cancer.