NCEH1 Human

What is the molecular structure and biochemical properties of human NCEH1?

Human NCEH1 (also known as AADACL1, KIAA1363) is a single, non-glycosylated polypeptide chain containing 298 amino acids (1-275 a.a.) with a molecular mass of 33.6 kDa . The protein belongs to the AB hydrolase superfamily .

The recombinant human NCEH1 is typically produced in E.coli expression systems and formulated in a buffer containing 20mM Tris-HCl (pH 8.0), 0.4M urea, and 10% glycerol . For optimal stability during research applications, the protein should be stored at 4°C if used within 2-4 weeks, or at -20°C for longer periods, with the addition of a carrier protein (0.1% HSA or BSA) recommended for long-term storage .

Methodologically, researchers working with NCEH1 should consider:

• Purification techniques: Affinity chromatography using His-tag fusion proteins achieves >90% purity as determined by SDS-PAGE

• Stability factors: Multiple freeze-thaw cycles significantly reduce enzyme activity

• Buffer composition: The presence of urea and glycerol in storage buffers maintains structural integrity

What are the validated methods for detecting and measuring NCEH1 expression?

Several complementary approaches can be employed to detect and quantify NCEH1 expression in experimental systems:

• Antibody-based detection: Polyclonal antibodies that detect endogenous levels of total NCEH1 protein are commercially available . These antibodies recognize both human and mouse NCEH1, making them valuable for comparative studies.

• Quantitative PCR: For mRNA expression analysis, validated primer sequences are:

  • Forward: 5′-AAGGTCTTCTCCGAAAGTGAAGG-3′

  • Reverse: 5′-CCTCCGTGGATATAGATGACGC-3′

  Expression should be normalized to appropriate housekeeping genes (e.g., β-actin) using the 2^(-ΔΔCt) method .

• Transcriptome analysis: RNA-sequencing data from databases such as TCGA and GTEx provide valuable resources for analyzing NCEH1 expression across different tissues and disease states .

When comparing NCEH1 expression between experimental groups, statistical approaches such as the Wilcoxon test are appropriate for assessing significance, particularly when comparing expression in pathological versus normal tissues .

How does NCEH1 interact with other proteins in lipid metabolism pathways?

NCEH1 functions within a network of interacting proteins involved in lipid metabolism and signaling. Key interaction partners include:

• Monoglyceride lipase (MGLL): This interaction suggests coordination in regulating lipid signaling molecules, with both enzymes belonging to the AB hydrolase superfamily .

• Liver carboxylesterase 1 (CES1): Functional connections between NCEH1 and CES1 reflect their shared roles in detoxification processes and lipid metabolism .

• Sterol O-acyltransferase 1 (SOAT1): This interaction is particularly relevant, as SOAT1 catalyzes the formation of fatty acid-cholesterol esters, while NCEH1 hydrolyzes these esters, indicating a potential regulatory feedback loop .

• E3 ubiquitin-protein ligase ZNRF1: In endothelial cells, NCEH1 interacts with ZNRF1, leading to the degradation of caveolin-1 (Cav-1) through the ubiquitination pathway .

Methodologically, these protein-protein interactions can be studied using co-immunoprecipitation followed by mass spectrometry, proximity ligation assays, and fluorescence resonance energy transfer (FRET) techniques.

How does NCEH1 contribute to atherosclerosis development?

NCEH1 plays a critical role in macrophage cholesterol metabolism, which has direct implications for atherosclerosis development:

• Functional mechanism: NCEH1 is responsible for cholesterol ester hydrolysis in macrophages, converting stored cholesterol esters into free cholesterol that can then be effluxed from the cell . This process is essential for reverse cholesterol transport and preventing foam cell formation.

• Pathophysiological significance: Impaired NCEH1 activity can lead to cholesterol ester accumulation in macrophages, promoting foam cell formation and contributing to atherosclerotic plaque development .

• Research approach: To study NCEH1's role in atherosclerosis, researchers typically employ:

  • Macrophage-specific NCEH1 knockout or overexpression models

  • High-fat diet-induced atherosclerosis models

  • Analysis of cholesterol ester/free cholesterol ratios in macrophages

  • Quantification of atherosclerotic lesion size and composition

The methodological challenges include distinguishing NCEH1 activity from other cholesterol ester hydrolases and accounting for compensatory mechanisms that may arise in genetic manipulation models.

What is the role of NCEH1 in endothelial dysfunction in diabetes?

NCEH1 has emerged as a protective factor against endothelial dysfunction in diabetes through several mechanisms:

• Expression pattern: NCEH1 expression and activity are reduced in high-fat diet (HFD)-induced mouse aortae, high glucose (HG)-exposed mouse aortae ex vivo, and HG-incubated primary endothelial cells .

• Functional significance: Endothelial-specific deficiency of NCEH1 exacerbates high glucose-induced impairment of endothelium-dependent relaxation (EDR), while NCEH1 overexpression restores impaired EDR .

• Molecular mechanism: NCEH1 ameliorates disrupted EDR by:

  • Dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1)

  • Promoting eNOS activation and nitric oxide (NO) release

  • Interacting with E3 ubiquitin-protein ligase ZNRF1 to facilitate Cav-1 degradation through the ubiquitination pathway

• Experimental evidence: Silencing Cav-1 and upregulating ZNRF1 improve EDR in diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolish the protective effects of NCEH1 .

Methodologically, these findings were established using:

• Endothelial-specific NCEH1 knockdown/overexpression via AAV5 vector injections under the control of a TIE1 promoter

• Ex vivo vessel tension measurements

• Protein interaction studies (co-immunoprecipitation)

• Ubiquitination assays

• Nitric oxide detection techniques

What is the significance of NCEH1 as a prognostic biomarker in pancreatic cancer?

Research using transcriptome data from TCGA and GTEx databases has revealed NCEH1 as a potential prognostic biomarker for pancreatic cancer:

The methodological approach included:

• Data mining from UCSC Xena browser

• Differential expression analysis between tumor and normal tissues

• Correlation analysis with clinicopathological features using Wilcoxon test

• Survival analysis using Kaplan-Meier curves

• Univariate and multivariate Cox regression

• Gene set enrichment analysis to identify associated pathways

How do post-translational modifications regulate NCEH1 activity?

While the search results don't provide explicit information on post-translational modifications of NCEH1, research methodologies to investigate this question would include:

• Identification of modification sites:

  • Mass spectrometry-based proteomics to identify phosphorylation, ubiquitination, and other modifications

  • Targeted mutagenesis of potential modification sites

  • Western blotting with modification-specific antibodies

• Functional consequences of modifications:

  • Enzyme activity assays comparing wild-type and mutant NCEH1

  • Protein stability assessments using cycloheximide (CHX) chase assays

  • Subcellular localization studies using fluorescence microscopy

• Regulatory enzymes:

  • Identification of kinases, phosphatases, and ubiquitin ligases that target NCEH1

  • Co-immunoprecipitation studies to confirm enzyme-substrate interactions

  • Inhibitor studies to validate regulatory relationships

The CHX chase assay methodology, as mentioned in the search results, provides a valuable approach for studying NCEH1 protein stability by treating cells with cycloheximide to inhibit protein synthesis and then following the degradation of existing protein over time .

What are the mechanisms by which NCEH1 promotes cancer cell migration and invasion?

NCEH1 has been implicated in cancer progression, particularly in promoting tumor cell migration . Research approaches to investigate the underlying mechanisms include:

• Cell-based assays:

  • Transwell migration and invasion assays with NCEH1-overexpressing or NCEH1-silenced cancer cells

  • Wound healing assays to assess collective cell migration

  • Live-cell imaging to track migration dynamics

• Molecular pathway analysis:

  • Gene expression profiling to identify downstream effectors

  • Focused studies on cell-cell adhesion molecules, given the association with cell-cell adhesion junctions in GSEA

  • Analysis of lipid mediators regulated by NCEH1 that may influence cancer cell behavior

• In vivo models:

  • Orthotopic xenograft models with modulated NCEH1 expression

  • Analysis of metastatic spread in relation to NCEH1 expression levels

  • Evaluation of tumor-stroma interactions

• Clinical correlation:

  • Analysis of NCEH1 expression in relation to invasion depth, lymph node metastasis, and distant metastasis

  • Correlation with epithelial-mesenchymal transition markers

  • Association with treatment response and patient outcomes

This multi-faceted approach would help elucidate the specific mechanisms by which NCEH1 promotes cancer progression, potentially identifying new therapeutic targets.

How can NCEH1-targeted therapies be developed for atherosclerosis and diabetes?

Based on NCEH1's roles in atherosclerosis and diabetes, several therapeutic development approaches can be considered:

• For atherosclerosis:

  • Small molecule activators of NCEH1 to enhance cholesterol ester hydrolysis in macrophages

  • Targeted delivery systems to increase NCEH1 expression specifically in arterial macrophages

  • Gene therapy approaches to overexpress NCEH1 in atherosclerotic plaques

• For diabetes-related endothelial dysfunction:

  • Compounds that stabilize or enhance NCEH1-ZNRF1 interaction

  • Agents that promote Cav-1 ubiquitination and degradation

  • Small molecules that disrupt the Cav-1/eNOS complex

• Methodological considerations for drug development:

  • High-throughput screening assays for NCEH1 activators

  • Structure-based drug design targeting NCEH1's active site

  • Evaluation of specificity to avoid off-target effects on related hydrolases

  • Assessment of tissue-specific effects

  • Pharmacokinetic and pharmacodynamic studies

• Biomarkers for patient stratification:

  • NCEH1 expression levels in accessible tissues or circulating cells

  • Genetic polymorphisms affecting NCEH1 expression or activity

  • Metabolic profiling to identify patients likely to benefit from NCEH1-targeted therapies

The development of such therapies would require interdisciplinary collaboration between structural biologists, medicinal chemists, pharmacologists, and clinicians specializing in cardiovascular and metabolic diseases.

What are the optimal in vivo models for studying NCEH1 function?

Several in vivo models have been developed or could be implemented to study NCEH1 function:

• Genetic models:

  • Global NCEH1 knockout mice

  • Tissue-specific NCEH1 knockout models (using Cre-loxP system)

  • Inducible knockout systems to avoid developmental compensation

  • Transgenic overexpression models

• AAV-mediated gene manipulation:

  • The TIE1 promoter-controlled AAV5 vector system has been successfully used for endothelial-specific manipulation of NCEH1 expression

  • This approach allows tissue-specific and temporal control of gene expression without germline modification

• Disease-specific models:

  • High-fat diet-induced atherosclerosis models

  • Streptozotocin-induced diabetes models

  • Orthotopic pancreatic cancer models

• Methodological considerations:

  • Confirmation of knockout/overexpression efficiency at both mRNA and protein levels

  • Assessment of compensatory mechanisms (e.g., upregulation of related hydrolases)

  • Careful selection of background strain to avoid strain-specific effects

  • Age and sex considerations in experimental design

• Readouts and analyses:

  • Enzyme activity assays in relevant tissues

  • Functional assessments (e.g., vessel reactivity studies for endothelial function)

  • Histological analysis of affected tissues

  • Lipidomic profiling to assess substrate and product levels

The choice of model should be guided by the specific research question, with consideration of the physiological relevance and limitations of each approach.

How can contradictory findings about NCEH1 function be reconciled in experimental design?

When faced with contradictory findings about NCEH1 function, researchers should consider several methodological approaches:

• Contextual differences:

  • Cell type specificity: NCEH1 may have different functions in macrophages versus endothelial cells

  • Disease context: The role of NCEH1 in cancer may differ from its role in metabolic diseases

  • Species differences: Human and mouse NCEH1 may have subtle functional differences

• Experimental design strategies:

  • Use multiple cell types and experimental systems to test hypotheses

  • Employ both in vitro and in vivo approaches

  • Conduct parallel studies in different disease models

  • Use both gain-of-function and loss-of-function approaches

• Technical considerations:

  • Validate antibody specificity thoroughly

  • Confirm gene knockdown/overexpression at both mRNA and protein levels

  • Use multiple methodologies to assess the same endpoint

  • Consider the impact of tags (e.g., His-tag) on protein function

• Data analysis approaches:

  • Meta-analysis of published studies

  • Pathway analysis to identify context-specific regulatory networks

  • Careful statistical analysis with appropriate controls for multiple comparisons

  • Consideration of effect sizes rather than just statistical significance

• Collaboration strategies:

  • Multi-laboratory validation studies

  • Exchange of reagents and protocols between research groups

  • Pre-registration of experimental designs to reduce publication bias

By systematically addressing these factors, researchers can better understand the context-dependent functions of NCEH1 and reconcile apparently contradictory findings.

What are the most promising future directions for NCEH1 research?

Based on current knowledge about NCEH1, several promising research directions emerge:

• Therapeutic development:

  • NCEH1 represents a promising candidate for the prevention and treatment of vascular complications of diabetes

  • Development of small molecule modulators of NCEH1 activity for atherosclerosis and cancer

  • Investigation of NCEH1 as a biomarker for patient stratification in pancreatic cancer

• Mechanistic studies:

  • Detailed structural studies of NCEH1 to facilitate drug design

  • Further investigation of the NCEH1-ZNRF1-Cav-1 pathway in different cellular contexts

  • Exploration of NCEH1's role in lipid mediator networks beyond cholesterol metabolism

• Translational research:

  • Validation of NCEH1 as a prognostic biomarker in larger patient cohorts

  • Development of diagnostic tests based on NCEH1 expression or activity

  • Clinical trials of NCEH1-targeted therapies in appropriate patient populations

• Emerging areas:

  • Role of NCEH1 in neurological disorders and neuroinflammation

  • Impact of NCEH1 on immune cell function and inflammatory responses

  • Influence of NCEH1 polymorphisms on disease susceptibility and progression

• Methodological advances:

  • Development of more specific and potent tools for manipulating NCEH1 in vivo

  • Single-cell analysis of NCEH1 expression and function in heterogeneous tissues

  • Advanced imaging techniques to visualize NCEH1 activity in real-time

The multifaceted roles of NCEH1 in lipid metabolism, signaling, and disease processes make it a fascinating target for continued research with significant potential for clinical translation.