Recombinant Bovine 3-hydroxyacyl-CoA dehydratase 4 (PTPLAD2)
Description
Role in Fatty Acid Synthesis
PTPLAD2 (HACD4) serves a crucial function in lipid metabolism, specifically in the elongation of very long-chain fatty acids (VLCFAs) . As a 3-hydroxyacyl-CoA dehydratase, it catalyzes the third reaction in the fatty acid elongation cycle, which involves the dehydration of 3-hydroxyacyl-CoA to 2,3-trans-enoyl-CoA . This enzymatic step is essential for the proper synthesis of VLCFAs, which are vital components of cellular membranes and various lipid-based signaling molecules.
The fatty acid elongation pathway consists of four sequential reactions:
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Condensation of malonyl-CoA with acyl-CoA to produce 3-ketoacyl-CoA (catalyzed by FA elongases)
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Reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA (catalyzed by 3-ketoacyl-CoA reductase)
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Dehydration of 3-hydroxyacyl-CoA to 2,3-trans-enoyl-CoA (catalyzed by HACD1-4, including PTPLAD2)
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Reduction to an acyl-CoA with two additional carbon chain units (catalyzed by 2,3-trans-enoyl-CoA reductase)
Molecular Interactions
The PTPLAD2 protein interacts with condensation enzymes of the ELOVL family, which are responsible for the first step in the fatty acid elongation cycle . This interaction suggests a coordinated mechanism for VLCFA synthesis, where the various enzymes involved in the pathway work in concert to ensure efficient lipid production. The HACD family, including PTPLAD2, was initially annotated with PTP-like nomenclature due to structural similarities to other proteins, but subsequent research has confirmed their true function in lipid metabolism .
Subcellular Localization
PTPLAD2 is primarily localized to the endoplasmic reticulum (ER) membrane, which is consistent with its role in fatty acid elongation . The ER serves as the primary site for lipid synthesis in eukaryotic cells, providing an ideal environment for the enzymatic activities involved in VLCFA production. The membrane-bound nature of PTPLAD2 facilitates its interaction with other components of the fatty acid elongation machinery.
Tissue Expression
In mammalian systems, PTPLAD2 exhibits a tissue-specific expression pattern. It is highly expressed in leukocytes, suggesting a potential role in immune function . Lower expression levels have been observed in heart, spleen, kidney, and placenta tissues . This differential expression across various tissues may reflect tissue-specific requirements for certain lipid species or specialized functions of PTPLAD2 in different cellular contexts.
Table 2: Tissue Expression Profile of PTPLAD2
| Tissue | Expression Level |
|---|---|
| Leukocytes | High |
| Heart | Low |
| Spleen | Low |
| Kidney | Low |
| Placenta | Low |
Recombinant Protein Production
Recombinant Bovine PTPLAD2 is typically produced in E. coli expression systems with an N-terminal His tag to facilitate purification . The protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol recommended for long-term storage . This recombinant protein serves as a valuable tool for studying the structure, function, and interactions of PTPLAD2 in various experimental settings.
Experimental Techniques
Various techniques are employed to study PTPLAD2 in research settings:
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Western Blot Analysis: Used to detect and quantify PTPLAD2 protein expression in cell and tissue samples. This typically involves protein extraction, SDS-PAGE separation, transfer to a membrane, and detection using specific antibodies .
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Plasmid Transfections: Overexpression studies using PTPLAD2-encoding plasmids can help elucidate the protein's function in cellular models. Lipofectamine 2000 is commonly used for transfection in cell culture systems .
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Gene Set Enrichment Analysis (GSEA): This computational method is used to identify biological pathways associated with PTPLAD2 expression or function, providing insights into the broader biological context of the protein's activity .
Role in Respiratory Diseases
Recent research has implicated PTPLAD2 in the pathogenesis of cigarette smoke-related conditions, particularly Chronic Obstructive Pulmonary Disease (COPD) . Studies have shown that cigarette smoke extract (CSE) significantly inhibits the expression of PTPLAD2 in human bronchial epithelial cells (BEAS-2B) in both concentration- and time-dependent manners . Furthermore, overexpression of PTPLAD2 has been found to mitigate CSE-induced decreases in cell viability, suggesting a potential protective role for this protein in respiratory health .
Signaling Pathway Involvement
Gene Set Enrichment Analysis has identified several signaling pathways potentially modulated by PTPLAD2, including:
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T-cell receptor signaling pathway
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Ribosomal function
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Intestinal immune network for IgA production
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Basal cell carcinoma pathways
These associations suggest that PTPLAD2 may have broader implications beyond lipid metabolism, potentially influencing immune function, cellular growth, and neurodegenerative processes.
Reconstitution Protocols
For optimal results when working with recombinant PTPLAD2, proper reconstitution is essential. The lyophilized protein should be briefly centrifuged before opening to bring contents to the bottom of the vial . Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, followed by the addition of glycerol (5-50% final concentration) for long-term storage . This preparation helps maintain protein stability and enzymatic activity.
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please indicate your desired format in the order notes, and we will prepare it accordingly.
Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please communicate this need in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is the basic structure and characteristics of PTPLAD2?
PTPLAD2 is a 232-amino acid membrane protein belonging to the protein tyrosine phosphatase-like A domain (PTPLAD) family. The protein contains conserved domains that contribute to its biological functions, particularly in signaling pathways. In humans, the PTPLAD2 gene maps to chromosome 9p21.3, a region frequently associated with tumor suppressor genes . The protein shows evolutionary conservation across multiple species, including chimpanzees, dogs, cows, mice, rats, and chickens, suggesting its fundamental biological importance across mammalian systems .
How is PTPLAD2 genetically conserved across species?
The genetic conservation of PTPLAD2 across multiple vertebrate species indicates its evolutionary significance. Research shows that PTPLAD2 maintains structural homology across chimpanzees, dogs, cows, mice, rats, and chickens . This high degree of conservation suggests that PTPLAD2 likely performs essential cellular functions that have been preserved throughout evolutionary history. Comparative genomic analyses can provide insights into functional domains that show the highest conservation, potentially identifying critical regions for protein function.
What are the recommended methods for PTPLAD2 expression analysis?
For accurate PTPLAD2 expression analysis, both transcriptional and translational detection methods are recommended:
RNA Expression Analysis:
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qRT-PCR using validated primers:
Protein Expression Analysis:
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Western blot analysis using:
How should experimental validation of PTPLAD2 overexpression be performed?
For overexpression studies, plasmid-based transfection is the established methodology:
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Obtain overexpression plasmids containing the complete coding sequence of PTPLAD2
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Include appropriate vector controls (e.g., pcDNA 3.1 vector as negative control)
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Seed cells at 2 × 10^5 cells/well in six-well plates
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Allow overnight attachment
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Perform transfection using standard protocols
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Validate expression using both qRT-PCR and Western blot
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Assess functional outcomes through appropriate assays (e.g., proliferation assays)
How is PTPLAD2 implicated in esophageal squamous cell carcinoma (ESCC)?
PTPLAD2 demonstrates significant tumor suppressor activity in ESCC:
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Expression patterns: ESCC tissue samples show loss of PTPLAD2 heterozygosity and decreased PTPLAD2 expression compared to normal tissue .
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Prognostic significance: Low PTPLAD2 expression correlates with poor patient prognosis in ESCC. This is frequently accompanied by high phosphorylated STAT3 (p-STAT3) levels .
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Functional validation: Overexpression of PTPLAD2 in ESCC cells results in:
These findings collectively establish PTPLAD2 as a potential tumor suppressor in ESCC carcinogenesis, operating primarily through modulation of the STAT3 signaling pathway.
What is the relationship between PTPLAD2 and chronic obstructive pulmonary disease (COPD)?
Research has identified PTPLAD2 as a potential biomarker in COPD pathogenesis:
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Expression alterations: PTPLAD2 expression is significantly reduced following cigarette smoke extract (CSE) treatment in BEAS-2B bronchial epithelial cells .
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Bioinformatic identification: PTPLAD2 was identified as one of the differentially expressed genes (DEGs) in COPD samples through analysis of the GSE76925 dataset .
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Pathway associations: PTPLAD2 may be involved in key COPD-related pathways including:
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Hub gene status: PTPLAD2 was identified as one of 14 hub genes through Weighted Gene Co-expression Network Analysis (WGCNA), underlining its central importance in COPD molecular networks .
How does PTPLAD2 modulate STAT3 signaling pathways?
PTPLAD2's tumor suppressor function appears to be mediated through STAT3 pathway modulation:
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Negative regulation: PTPLAD2 overexpression reduces STAT3 phosphorylation levels, suggesting it may function as a negative regulator of STAT3 activation .
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Downstream effects: By reducing STAT3 phosphorylation, PTPLAD2 decreases expression of FoxM1, a transcription factor regulated by STAT3 that promotes cell proliferation .
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Functional consequences: The inhibition of STAT3 signaling by PTPLAD2 results in suppressed cell proliferation and reduced tumor formation capacity .
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Clinical correlation: The inverse relationship between PTPLAD2 expression and p-STAT3 levels in patient samples supports this regulatory mechanism in vivo .
What other signaling pathways interact with PTPLAD2?
Beyond STAT3 signaling, bioinformatic analyses suggest PTPLAD2 involvement in additional pathways:
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PI3K-Akt pathway: Gene Ontology and KEGG pathway analyses indicate PTPLAD2 may interact with the phosphoinositide 3-kinase-Akt signaling pathway, critical for cell survival and proliferation .
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ECM-receptor interaction: PTPLAD2 appears to be involved in pathways related to extracellular matrix interactions, potentially influencing cell adhesion and migration .
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mRNA processing: Bioinformatic evidence suggests PTPLAD2 may play a role in mRNA processing and viral transcription pathways .
What controls should be included in PTPLAD2 functional studies?
Robust experimental design for PTPLAD2 studies should include:
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Expression controls:
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Technical controls:
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Validation approaches:
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Multiple detection methods (qRT-PCR and Western blot)
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Multiple cell lines to confirm findings
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Rescue experiments to confirm specificity of effects
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How should cellular models be selected for PTPLAD2 research?
Model selection should be guided by:
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Disease relevance:
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Expression characteristics:
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Consider baseline PTPLAD2 expression levels
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Verify chromosome 9p21.3 status, particularly for cancer cell lines
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Assess STAT3 pathway activity
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Functional readouts:
How might PTPLAD2 serve as a biomarker or therapeutic target?
PTPLAD2 shows significant potential as both a biomarker and therapeutic target:
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Diagnostic and prognostic biomarker:
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Therapeutic targeting strategies:
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Restoring PTPLAD2 expression could suppress STAT3 hyperactivation in cancers
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Targeting the PTPLAD2-STAT3-FoxM1 axis represents a potential intervention point
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Combination approaches targeting PTPLAD2 and related pathways may enhance efficacy
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Considerations for implementation:
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Tissue-specific expression patterns must be considered
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Delivery methods for PTPLAD2-based therapeutics require optimization
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Potential off-target effects need thorough evaluation
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What are the current limitations in PTPLAD2 research?
Despite promising findings, several challenges remain:
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Mechanistic understanding:
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The precise molecular mechanism by which PTPLAD2 inhibits STAT3 phosphorylation remains unclear
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Comprehensive protein interaction networks for PTPLAD2 have not been fully elucidated
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The relationship between PTPLAD2's membrane localization and its function needs further investigation
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Technical challenges:
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Standardized antibodies and detection methods across studies are lacking
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In vivo models for PTPLAD2 function assessment require further validation
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High-throughput screening approaches for PTPLAD2 modulators need development
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Translational gaps:
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The clinical relevance of PTPLAD2 requires validation in larger patient cohorts
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Delivery methods for PTPLAD2-based therapeutics remain underdeveloped
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Potential compensatory mechanisms that might limit therapeutic efficacy need exploration
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