PPA1 Human

What is the basic structure of human PPA1?

Human PPA1 is a 289-amino acid protein with a molecular weight of approximately 42 kDa. The crystal structure has been resolved at 2.4 Å resolution, revealing that PPA1 forms a dimeric structure that is conserved across animal and fungal PPases . The monomeric protein fold is similar to those found in other family I PPases, with a largely pre-organized and relatively rigid active site designed for pyrophosphate hydrolysis . The active site can accommodate both inorganic pyrophosphate and phosphorylated peptides such as those from JNK1 .

What is the primary catalytic function of PPA1?

PPA1 primarily catalyzes the hydrolysis of pyrophosphate (PPi) to two phosphate ions, a highly exergonic reaction that is utilized in many biochemical pathways . This reaction is essential for making processes like DNA synthesis and bone formation effectively irreversible . PPA1 can also be coupled to unfavorable biochemical reactions to drive them to completion, such as in the synthesis of activated sulfur donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) . Beyond its canonical function, PPA1 can directly dephosphorylate phosphorylated c-Jun N-terminal kinases 1 (JNK1) .

How is PPA1 different from PPA2?

Humans possess two inorganic pyrophosphatases: PPA1 and PPA2. While both catalyze the hydrolysis of pyrophosphate, PPA1 is ubiquitously expressed in all tissues and acts largely as a housekeeping enzyme with high activity on pyrophosphate . PPA2 has a more restricted expression pattern. PPA1 has emerged as particularly significant in cancer research due to its upregulation in various malignancies and its additional role in directly dephosphorylating JNK1 .

What are the established protocols for measuring PPA1 enzymatic activity?

The enzymatic activity of recombinant human PPA1 can be measured using the following protocol:

• Prepare a substrate solution of 16 mM Sodium Pyrophosphate in deionized water

• Dilute rhPPA1 to 0.4 μg/mL in assay buffer

• Combine 25 μL of substrate with 25 μL of diluted rhPPA1 (create a substrate blank by combining substrate with assay buffer)

• Incubate the reaction and blank for 10 minutes at room temperature

• Measure phosphate production using a Malachite Green Phosphate Detection Kit

• Quantify results using a plate reader at appropriate wavelength

This assay allows for the precise measurement of PPA1's ability to hydrolyze pyrophosphate under controlled conditions. For investigating PPA1's protein phosphatase activity on JNK1, modified protocols using phosphorylated peptide substrates are recommended.

What expression systems are most effective for producing recombinant PPA1 for structural and functional studies?

E. coli expression systems have proven effective for producing recombinant human PPA1 for structural and functional studies. The standard approach uses a construct encoding Met1-Asn289 of human PPA1 with a C-terminal 6-His tag for purification . For carrier-free preparations needed in certain applications, the protein should be supplied in a buffer containing Tris, NaCl, glycerol, and DTT without bovine serum albumin (BSA) . When BSA might interfere with downstream applications, carrier-free versions are recommended, while BSA-containing preparations are preferred for cell culture applications or as ELISA standards due to enhanced stability and shelf-life .

What methods are recommended for investigating PPA1-protein interactions, particularly with JNK1?

For investigating PPA1-protein interactions with JNK1 and other potential binding partners, researchers should consider:

• Co-immunoprecipitation (Co-IP): Use anti-PPA1 antibodies to pull down protein complexes from cell lysates, followed by Western blot analysis for JNK1 or other suspected interacting proteins.

• Bioluminescence Resonance Energy Transfer (BRET): Tag PPA1 and potential binding partners with appropriate donor and acceptor proteins to detect real-time interactions in living cells.

• Molecular modeling: As demonstrated in the literature, computational modeling of PPA1 in complex with JNK1-derived phosphopeptides can provide insights into the molecular basis of their interaction .

• Mutagenesis studies: Create site-directed mutations in the active site of PPA1 to identify residues critical for JNK1 binding and dephosphorylation.

These approaches enable researchers to characterize the direct interaction between PPA1 and JNK1, which is crucial for understanding PPA1's role in cancer progression beyond its canonical pyrophosphatase activity .

How is PPA1 expression altered in different cancer types?

PPA1 expression is significantly increased in multiple cancer types compared to corresponding normal tissues:

The level of PPA1 expression correlates with clinicopathological staging in several cancers, with higher grades and stages showing higher PPA1 expression. This pattern has been particularly noted in gastric cancer, epithelial ovarian cancer, and colorectal cancer .

What molecular mechanisms underlie PPA1's role in cancer progression?

PPA1 contributes to cancer progression through several molecular mechanisms:

• Cell proliferation and apoptosis regulation:

  • Silencing PPA1 reduces proliferation and promotes apoptosis in lung and breast cancer cells

  • This effect is associated with increased expression of p21, p53, and cleaved caspase-3, and decreased expression of Ki-67

  • The role appears to be p53-dependent, as confirmed in TP53-deficient H1299 cells where PPA1 manipulation had no effect

• JNK pathway modulation:

  • PPA1 promotes dephosphorylation of p-JNK1 at the peptide level

  • This inhibits apoptosis in lung cancer cells

  • In colorectal cancer, PPA1 upregulation promotes dephosphorylation of p-JNK1 without affecting p-ERK or p-p38 levels

• EMT and metastasis promotion:

  • PPA1 participates in epithelial-mesenchymal transition (EMT)-related signaling pathways

  • This promotes tumor metastasis in various cancers

• Energy metabolism:

  • As an energy metabolism-related enzyme, PPA1 maintains cellular metabolism in mitochondria and the expression of the key metabolite NAD+

These mechanisms collectively contribute to enhanced tumor cell survival, proliferation, and metastatic potential .

What is the potential of PPA1 as a prognostic biomarker and therapeutic target?

PPA1 shows significant promise as both a prognostic biomarker and therapeutic target:

As a prognostic biomarker:

As a therapeutic target:

• PPA1 knockdown induces apoptosis and decreases proliferation in cancer cells

• Several approaches to targeting PPA1 show promise:

  • Developing small-molecule inhibitors specifically targeting PPA1

  • Exploring miRNAs to regulate PPA1 expression (e.g., miR-545-3p represses PPA1 mRNA expression)

  • Combining PPA1 inhibition with JNK or PI3K-AKT pathway inhibitors (such as JNK-IN-8, BKM120, or Capivasertib)

  • Targeting PPA1 to curb metabolic plasticity of tumors, either as standalone therapy or in combination with chemotherapy

The therapeutic potential is supported by the observation that PPA1 knockdown reduces malignant behaviors including tumor proliferation and migration .

How does PPA1 interact with cellular signaling pathways beyond JNK?

While the interaction with JNK is well-documented, PPA1 also influences other signaling pathways:

• Wnt/β-catenin pathway:

  • PPA1 has been implicated in modulating this pathway, which is crucial for cancer cell stemness and proliferation

• PI3K/AKT/GSK-3β signaling:

  • PPA1 regulates tumor cytogenesis development through this pathway, which is central to cell survival and proliferation

• p53 pathway:

  • PPA1's effects on apoptosis appear to be intricately linked to p53 function

  • In p53-deficient cells, PPA1 manipulation does not affect proliferation or apoptosis

• Cellular metabolism:

  • PPA1 maintains cellular metabolism in mitochondria

  • It affects the expression of key metabolites like NAD+, suggesting a role in metabolic reprogramming of cancer cells

These interactions highlight PPA1's role as a multifunctional regulator at the intersection of energy metabolism and signaling pathways that control cell fate decisions.

What is known about the transcriptional and post-transcriptional regulation of PPA1?

Transcriptional regulation:

• The PPA1 promoter contains three putative Sp1 binding sites, with one exhibiting the highest transcriptional activity

• Sp1, a constitutive transcription factor often overexpressed in cancers, activates PPA1 promoter activity, upregulates protein expression, and increases chromatin accessibility

• Histone acetyltransferase (p300) further activates Sp1-induced PPA1 promoter activity

• The PPA1 promoter may undergo local chromatin remodeling through histone acetylation/deacetylation processes

Post-transcriptional regulation:

• miR-545-3p represses PPA1 mRNA expression

• circ_0067934 acts as a molecular sponge for miR-545-3p, thereby promoting PPA1 expression

• Additional miRNAs likely regulate PPA1 expression and could be therapeutically relevant targets

This multilayered regulation suggests that PPA1 expression can be modulated at multiple levels, providing diverse opportunities for therapeutic intervention.

What structural features of PPA1 determine its substrate specificity for both pyrophosphate and phosphorylated proteins?

The crystal structure of human PPA1 at 2.4 Å resolution reveals important insights into its dual substrate specificity:

• Active site architecture:

  • PPA1 has a largely pre-organized and relatively rigid active site optimized for pyrophosphate hydrolysis

  • The active site contains metal-binding residues that coordinate metal ions (typically Mg2+) essential for catalysis

• Accommodation of phosphorylated peptides:

  • Modeling studies indicate that the active site of PPA1 can accommodate double-phosphorylated peptides from JNK1

  • This structural flexibility allows PPA1 to function both as a pyrophosphatase and a protein phosphatase

• Dimeric structure:

  • PPA1 forms a dimeric structure that is conserved across animal and fungal PPases

  • This quaternary structure may influence substrate recognition and catalytic efficiency

• Conserved catalytic residues:

  • Key catalytic residues are positioned to facilitate nucleophilic attack on the phosphorus atom

  • These residues are conserved across family I PPases but may be arranged slightly differently to accommodate both inorganic and protein substrates

These structural features provide the molecular basis for PPA1's dual functionality and offer potential targets for structure-based drug design.

What are the main challenges in developing specific inhibitors for PPA1?

Developing specific inhibitors for PPA1 presents several challenges:

• Structural similarity with other phosphatases:

  • PPA1 shares structural features with other phosphatases, making it difficult to achieve selectivity

  • Cross-reactivity with other essential phosphatases could lead to significant off-target effects

• Dual substrate specificity:

  • PPA1's ability to act on both pyrophosphate and phosphorylated proteins complicates inhibitor design

  • Inhibitors may need to be tailored to block specific activities while preserving others, depending on the therapeutic goal

• Essential housekeeping function:

  • As PPA1 is ubiquitously expressed and serves essential housekeeping functions, complete inhibition could cause widespread cellular toxicity

  • Developing inhibitors with cancer-cell specificity is crucial

• Active site characteristics:

  • The pre-organized and relatively rigid active site of PPA1 may limit the binding of bulky inhibitor molecules

  • The metal-dependent catalytic mechanism adds complexity to inhibitor design

• Limited structural data on human PPA1-inhibitor complexes:

  • Despite the crystal structure being available, more detailed studies of PPA1 in complex with various inhibitors are needed to guide rational drug design

These challenges necessitate sophisticated approaches to inhibitor design, potentially including fragment-based drug discovery, structure-based virtual screening, and allosteric inhibitor development.

How can researchers effectively distinguish between PPA1's enzymatic and non-enzymatic functions in complex cellular environments?

Differentiating between PPA1's enzymatic and non-enzymatic functions requires sophisticated experimental approaches:

• Catalytically inactive mutants:

  • Generate PPA1 mutants with point mutations in catalytic residues that abolish enzymatic activity while preserving protein structure

  • Compare the effects of wild-type and catalytically inactive PPA1 on cellular processes to identify functions independent of enzymatic activity

• Domain-specific antibodies:

  • Develop antibodies that selectively block either the active site or potential protein-interaction domains

  • Use these antibodies to selectively inhibit specific functions of PPA1

• Substrate-specific activity assays:

  • Develop assays that specifically measure PPA1's activity on different substrates (pyrophosphate versus phosphorylated JNK1)

  • This allows researchers to correlate specific enzymatic activities with observed cellular phenotypes

• Proximity labeling techniques:

  • Use BioID or APEX2 proximity labeling fused to PPA1 to identify proteins in close proximity

  • This can reveal potential non-enzymatic interaction partners and functions

• Domain-swapping experiments:

  • Create chimeric proteins where domains of PPA1 are swapped with corresponding domains from related proteins

  • This can help identify regions responsible for specific functions

• Selective small molecule inhibitors:

  • Once developed, inhibitors that selectively block pyrophosphatase activity versus protein phosphatase activity would be valuable tools for dissecting PPA1's functions

These approaches, used in combination, can help delineate the complex roles of PPA1 in cellular processes and disease progression.

What are the recommended approaches for studying PPA1 in the context of the tumor microenvironment?

Studying PPA1 in the tumor microenvironment requires specialized methodologies:

• 3D organoid cultures:

  • Develop patient-derived organoids that recapitulate the cellular heterogeneity of tumors

  • Manipulate PPA1 expression in specific cell types within the organoid to assess effects on tumor-stromal interactions

• Co-culture systems:

  • Establish co-cultures of cancer cells with stromal cells, immune cells, or endothelial cells

  • Assess how PPA1 manipulation in cancer cells affects surrounding non-cancer cells and vice versa

• Metabolic profiling:

  • As PPA1 is involved in energy metabolism, conduct comprehensive metabolomics analyses

  • Focus on how PPA1 expression alters metabolite exchange between cancer cells and stromal cells

• In vivo models with cell-type specific manipulation:

  • Generate mouse models with cell-type specific knockout or overexpression of PPA1

  • This allows assessment of PPA1's role in specific compartments of the tumor microenvironment

• Spatial transcriptomics and proteomics:

  • Use spatial profiling techniques to map PPA1 expression and activity across different regions of the tumor

  • Correlate with markers of hypoxia, inflammation, and immune infiltration

• Ex vivo tissue slice cultures:

  • Maintain tumor tissue slices in culture to preserve tissue architecture and cellular interactions

  • Test PPA1 inhibitors or genetic manipulation in this context to better predict in vivo responses

These approaches acknowledge that PPA1's role in cancer likely extends beyond cancer cell-autonomous effects to include modulation of the tumor microenvironment and metabolic interactions between different cell types .

What emerging technologies might advance PPA1 research and therapeutic development?

Several emerging technologies hold promise for advancing PPA1 research:

• CRISPR-based screening approaches:

  • Genome-wide CRISPR screens to identify synthetic lethal interactions with PPA1 inhibition

  • CRISPR activation/inhibition screens to identify genes that modulate sensitivity to PPA1 targeting

• Single-cell multi-omics:

  • Single-cell RNA sequencing combined with proteomics to identify cell populations most dependent on PPA1

  • This could reveal cancer cell subpopulations particularly vulnerable to PPA1 inhibition

• AI-driven drug discovery:

  • Machine learning approaches to predict novel PPA1 inhibitors based on structural data

  • Deep learning models to optimize inhibitor design for specificity and efficacy

• Nanobody and aptamer development:

  • Development of nanobodies or aptamers targeting PPA1 for both research and therapeutic applications

  • These smaller molecules may access structural features not easily targeted by conventional antibodies

• Targeted protein degradation:

  • PROTACs (Proteolysis Targeting Chimeras) or molecular glues to selectively degrade PPA1

  • This approach may overcome limitations of catalytic inhibition

• In situ structural biology:

  • Cryo-electron tomography to visualize PPA1 complexes in their native cellular environment

  • This could reveal previously unknown interaction partners and functions

These technologies could accelerate the development of PPA1-targeted therapies and deepen our understanding of its roles in normal physiology and disease.

How can researchers better translate PPA1 findings from basic research to clinical applications?

To effectively translate PPA1 research into clinical applications, researchers should consider:

• Development of robust biomarkers:

  • Standardize methods for measuring PPA1 expression and activity in patient samples

  • Develop companion diagnostics to identify patients most likely to benefit from PPA1-targeted therapies

• Patient-derived models:

  • Establish patient-derived xenografts and organoids to test PPA1 inhibition in personalized models

  • Correlate response with molecular features to identify predictive biomarkers

• Combination therapy approaches:

  • Systematically test PPA1 inhibition in combination with standard-of-care treatments

  • Focus on potential synergies with JNK or PI3K-AKT pathway inhibitors as suggested by molecular studies

• Animal models that recapitulate PPA1 dysregulation:

  • Develop genetically engineered mouse models with tissue-specific PPA1 overexpression

  • Use these to test prevention and intervention strategies

• Early engagement with clinical and regulatory stakeholders:

  • Identify and address potential challenges in clinical development early

  • Consider innovative clinical trial designs appropriate for targeted therapies

• Focus on cancer types with strongest PPA1 association:

  • Prioritize clinical development in cancers with established PPA1 upregulation and correlation with poor outcomes

  • This includes lung, breast, colorectal, and gastric cancers

These translational approaches can help bridge the gap between promising preclinical findings and effective clinical applications of PPA1-targeted therapies.

What are the potential implications of targeting PPA1 for cancer metabolism and treatment resistance?

Targeting PPA1 could have significant implications for cancer metabolism and treatment resistance:

• Metabolic vulnerabilities:

  • PPA1 inhibition may create metabolic vulnerabilities by disrupting pyrophosphate homeostasis

  • This could synergize with other metabolism-targeting therapies, such as glycolysis inhibitors

• NAD+ metabolism:

  • PPA1 maintains the expression of key metabolite NAD+

  • Inhibition could disrupt NAD+-dependent processes, including DNA repair, potentially sensitizing cells to DNA-damaging agents

• Resistance mechanisms:

  • Potential resistance mechanisms to PPA1 inhibition should be anticipated

  • These might include upregulation of PPA2, activation of alternative phosphatases, or metabolic rewiring

• Microenvironmental interactions:

  • PPA1 inhibition may alter metabolic interactions within the tumor microenvironment

  • This could affect immune cell function and tumor-stromal cross-talk

• Combinatorial approaches:

  • Rational combinations with therapies targeting complementary pathways

  • Particularly promising are combinations with JNK or PI3K-AKT pathway inhibitors

• Biomarkers of response:

  • Metabolic signatures that predict response to PPA1 inhibition

  • These could include pyrophosphate levels, JNK phosphorylation status, or downstream metabolic alterations

Understanding these implications will be crucial for developing effective PPA1-targeted therapies and anticipating potential resistance mechanisms before they emerge in clinical settings .