NSDHL Human

What is the basic molecular structure of human NSDHL protein?

Human NSDHL is a membrane-anchored enzyme that functions in cholesterol biosynthesis. Recent crystal structure determinations have revealed that NSDHL contains a coenzyme-binding site that undergoes conformational changes upon NAD+ binding. X-ray crystallography has provided two crystal structures (NSDHL apo and NSDHL holo) that reveal atomic-level details of the enzyme's architecture . Structurally, the truncated construct (amino acids 31-267) of the full sequence (1-373) has been instrumental in crystallization studies. The protein exists in a conformational state that changes upon coenzyme binding, providing critical insight into the enzyme's catalytic mechanism .

How does NSDHL participate in the cholesterol biosynthesis pathway?

NSDHL catalyzes a critical step in post-squalene cholesterol biosynthesis. Specifically, it participates in the conversion of lanosterol to cholesterol by facilitating the removal of a methyl group (one carbon atom and three hydrogen atoms) from lanosterol . The enzyme functions as part of a complex that performs NAD+-dependent oxidative decarboxylation of the C4 methyl groups of 4α-carboxysterols . This reaction occurs on the endoplasmic reticulum (ER) membrane, where NSDHL is primarily localized, although it can also be found in lipid droplets, which are ER-derived cytoplasmic structures for storing lipids and cholesterols .

What experimental approaches can determine NSDHL oligomerization state?

Size-exclusion chromatography with multiangle light scattering (SEC-MALS) has been successfully employed to determine the oligomeric state of NSDHL. In published research, SEC-MALS experiments for both NSDHL apo and NSDHL holo forms utilized a fast protein liquid chromatography (FPLC) system connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer . The methodology involves:

• Pre-equilibration of a Superdex 200 10/300 GL gel filtration column with an appropriate buffer (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 8.0)

• Normalization using ovalbumin protein

• Injection of the purified protein (5 mg/ml) at a flow rate of 0.4 ml/min

• Data analysis using the Zimm model for fitting static light scattering data and graphing using ASTRA 6 software

This approach reveals crucial information about NSDHL's biologically relevant quaternary structure, which is essential for understanding its enzymatic function.

What are the developmental expression patterns of NSDHL in mammalian tissues?

NSDHL shows distinct tissue-specific expression patterns during embryonic and postnatal development. Immunohistochemistry studies in mice have revealed that the highest levels of NSDHL expression during embryogenesis occur in:

• Liver

• Dorsal root ganglia

• Central nervous system

• Retina

• Adrenal gland

• Testis

Postnatally, NSDHL expression patterns shift, with particularly high expression in cerebral cortical and hippocampal neurons . The developmental regulation of NSDHL expression suggests critical roles for cholesterol biosynthesis during specific stages of organogenesis and tissue maturation. Researchers investigating NSDHL should consider these tissue-specific patterns when designing experiments and interpreting results in developmental contexts.

How do mutations in NSDHL cause CHILD syndrome, and what methodologies are used to study genotype-phenotype correlations?

Mutations in the X-linked NSDHL gene cause CHILD (Congenital Hemidysplasia with Ichthyosiform nevus and Limb Defects) syndrome in humans . Several methodological approaches have been used to understand genotype-phenotype correlations:

• Genetic sequencing to identify specific NSDHL variants - most variants change single amino acids in the enzyme, while others delete part or all of the gene

• Functional assays to determine how mutations affect enzyme activity, with many preventing production of functional NSDHL enzyme

• Mouse models carrying mutant alleles of Nsdhl (e.g., bare patches/Bpa mice) that recapitulate aspects of the human condition

• Microarray analysis comparing gene expression in cells expressing mutant versus wild-type NSDHL

• Immunohistochemistry in heterozygous Bpa females, which are mosaic for NSDHL expression due to random X-inactivation

The unilateral distribution of symptoms in CHILD syndrome likely reflects the pattern of X-chromosome inactivation, with affected tissues showing predominant expression of the mutant allele. Researchers have observed that NSDHL-deficient cells can survive and differentiate during embryonic development but appear to be subject to negative selection over time, as evidenced by declining proportions of NSDHL-negative cells in the liver and brain of Bpa mice during the first year of life .

What methodologies are optimal for assessing the impact of NSDHL deficiency on embryonic development?

To study the impact of NSDHL deficiency on embryonic development, researchers have employed several complementary methodologies:

• Mouse models: The Bpa1H allele of Nsdhl, defined by a K103X nonsense mutation, has been used to study developmental abnormalities. Heterozygous Bpa1H females display skin and skeletal abnormalities reflecting random X-inactivation patterns, while hemizygous males die before embryonic day 10.5 .

• Immunohistochemistry: This technique allows visualization of NSDHL expression in tissue sections and identification of NSDHL-deficient cells in mosaic females. In Bpa heterozygous female embryos, researchers have detected NSDHL-deficient cells in developing cerebral cortex and retina .

• Microarray analysis: Comparing gene expression profiles between embryonic fibroblasts expressing the Bpa1H allele versus wild-type cells provides insights into molecular pathways disrupted by NSDHL deficiency .

• Lineage tracing: Tracking the fate of NSDHL-deficient cells throughout development reveals their ability to survive and differentiate during embryogenesis, despite being subject to negative selection postnatally .

These approaches collectively enable researchers to elucidate how disruption of cholesterol biosynthesis affects specific developmental processes and how compensatory mechanisms might operate in mosaic tissues.

What is the relationship between NSDHL and EGFR signaling in cancer cells?

NSDHL plays a significant role in regulating epidermal growth factor receptor (EGFR) expression and signaling in cancer cells. Research has shown that:

• NSDHL regulates EGFR trafficking pathways, affecting receptor availability at the cell surface

• Loss of NSDHL gene expression can sensitize cancer cells to EGFR-targeting inhibitors

• NSDHL deficiency leads to accumulation of sterol metabolites that can suppress tumor growth

These findings suggest that NSDHL represents a potential therapeutic target in EGFR-driven cancers. Researchers investigating this relationship should design experiments that examine both cholesterol metabolism and EGFR signaling pathways simultaneously to fully understand the interactions between these systems.

How can researchers develop and evaluate NSDHL inhibitors as potential cancer therapeutics?

The development and evaluation of NSDHL inhibitors follows a structured methodological approach:

• Structure-based virtual screening: Using crystal structures of NSDHL (e.g., NAD+-bound NSDHL, PDB code: 6JKH) to screen molecular libraries. The receptor grid box is typically generated with a 30 Å × 30 Å × 30 Å cube centered on complexed NAD+ .

• Biochemical evaluation of potential inhibitors: Competitive inhibitor assays performed in 96-well flat-bottom black microplates with library compounds at 100 μM, NSDHL at 32 μM in assay buffer, and NADH at 80 μM. Fluorescence intensity measurements (Ex/Em = 340/460 nm) allow calculation of inhibition percentages .

• Cell-based assays: Testing the effects of NSDHL inhibitors on EGFR protein turnover and signaling cascades in cancer cell lines.

• Combination studies: Evaluating the ability of NSDHL inhibitors to sensitize cancer cells to established therapies, such as EGFR kinase inhibitors like erlotinib, in both drug-sensitive and drug-resistant cell lines .

One successful application of this approach identified compound 9 with an IC50 of approximately 8 μM, which altered EGFR protein turnover, suppressed EGFR signaling, and enhanced sensitivity to erlotinib in cancer cells .

What experimental approaches can differentiate between the cholesterol-dependent and cholesterol-independent functions of NSDHL in cancer?

Differentiating between cholesterol-dependent and cholesterol-independent functions of NSDHL in cancer requires sophisticated experimental designs:

• Genetic manipulation combined with metabolite supplementation: Knockdown or knockout of NSDHL followed by supplementation with downstream cholesterol metabolites can help determine whether phenotypic effects are due to cholesterol deficiency or other NSDHL functions.

• Specific inhibitors: Comparing the effects of NSDHL inhibitors (targeting enzymatic activity) with those of other cholesterol synthesis pathway inhibitors can identify NSDHL-specific effects beyond cholesterol production.

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screens can identify NSDHL interaction partners unrelated to cholesterol synthesis.

• Subcellular localization analyses: Since NSDHL localizes to both the ER membrane and lipid droplets , examining the functional consequences of disrupting localization to specific compartments can reveal compartment-specific roles.

• Pathway analysis: Microarray or RNA-seq comparison of gene expression changes induced by NSDHL deficiency versus other cholesterol synthesis enzyme deficiencies can identify unique NSDHL-regulated pathways.

These approaches collectively allow researchers to dissect the multifaceted roles of NSDHL in cancer biology beyond its canonical function in cholesterol biosynthesis.

What are the optimal conditions for expressing and purifying recombinant human NSDHL for structural studies?

Based on successful crystallization studies, the optimal approach for expressing and purifying recombinant human NSDHL involves:

• Construct design: Using a truncated construct (amino acids 31-267) of the full sequence (1-373) to improve solubility and crystallization properties .

• Expression system: Cloning the NSDHL sequence into pET21a vector (or similar) and overexpressing in E. coli after verifying DNA sequences .

• Purification strategy: A multi-step purification process typically including:

  • Initial capture by affinity chromatography

  • Buffer exchange and further purification steps

  • Quality control by SDS-PAGE and activity assays

• Buffer optimization: For crystallization studies, determining optimal buffer conditions that maintain protein stability while promoting crystal formation.

This approach has successfully yielded protein suitable for X-ray crystallography, enabling determination of both apo and holo structures of human NSDHL .

What methods can assess the stability and activity of wild-type versus mutant NSDHL proteins?

Several complementary methodologies can evaluate the stability and activity of NSDHL proteins:

• Thermal shift assay (TSA): This technique measures protein thermal stability and can detect differences between wild-type and mutant proteins. The protocol involves:

  • Diluting protein solutions to 5 μM in an appropriate buffer

  • Adding SYPRO Orange as a fluorescent reporter

  • Ramping temperature from 25 to 95°C at 1°C/min

  • Measuring fluorescence using real-time PCR equipment

  • Calculating midpoint melting temperature (Tm) values using Boltzmann sigmoidal fitting

• Isothermal titration calorimetry (ITC): This approach measures binding affinities between NSDHL and its cofactors (NAD+, NADH, NADP+, NADPH). The methodology involves multiple injections of cofactor into the protein solution while measuring heat changes, allowing determination of binding constant (Kd), enthalpy change (ΔH), and binding stoichiometry (N) .

• Enzymatic activity assays: Fluorescence-based assays measuring NADH utilization or product formation can assess catalytic function of wild-type versus mutant NSDHL proteins.

• Protein stability in cells: Western blotting after cycloheximide chase can determine protein half-life differences between wild-type and mutant NSDHL in cellular contexts.

These methods provide comprehensive characterization of how mutations affect NSDHL structure, stability, cofactor binding, and catalytic function.

How can researchers most effectively analyze NSDHL expression patterns in different tissues and developmental stages?

Effective analysis of NSDHL expression patterns across tissues and developmental stages requires a multi-faceted approach:

• Immunohistochemistry (IHC): This technique has been successfully used to analyze NSDHL expression in embryonic and postnatal tissues. IHC allows visualization of protein expression at the cellular level, revealing tissue-specific patterns and subcellular localization .

• Quantitative PCR (qPCR): For quantitative assessment of NSDHL mRNA levels across tissues and developmental timepoints.

• Western blotting: To determine NSDHL protein levels in tissue lysates, providing quantitative comparison across samples.

• Single-cell RNA sequencing: For high-resolution analysis of NSDHL expression in heterogeneous tissues, revealing cell type-specific expression patterns.

• Reporter gene constructs: Creating transgenic models with NSDHL promoter driving reporter gene expression allows visualization of expression patterns in live tissues.

• Temporal analysis: For developmental studies, sampling at multiple timepoints is crucial. In mice, analysis of expression from embryonic stages through postnatal day 6 and into adulthood (up to one year) has revealed dynamic changes in NSDHL expression patterns and the fate of NSDHL-deficient cells in mosaic females .

• X-chromosome inactivation analysis: In heterozygous females (e.g., Bpa mouse models), the mosaic pattern of NSDHL expression due to random X-inactivation can be leveraged to study cell-autonomous effects of NSDHL deficiency .

These complementary approaches provide a comprehensive picture of NSDHL expression across tissues and developmental stages, informing hypotheses about its tissue-specific functions.

How might single-cell analysis techniques advance our understanding of NSDHL function in heterogeneous tissues?

Single-cell analysis techniques offer unprecedented opportunities to dissect NSDHL function in complex tissues:

• Single-cell RNA sequencing (scRNA-seq): This methodology can reveal cell type-specific expression patterns of NSDHL and identify compensatory pathways activated in different cell populations in response to NSDHL deficiency. Particularly valuable for studying heterozygous females with mosaic expression patterns due to X-inactivation.

• Single-cell proteomics: Emerging techniques for protein analysis at single-cell resolution could detect differences in NSDHL protein levels and post-translational modifications across cell types.

• Spatial transcriptomics/proteomics: These approaches preserve spatial information while providing transcriptomic or proteomic data, potentially revealing microenvironmental influences on NSDHL expression and function.

• Single-cell metabolomics: Techniques for measuring metabolites in individual cells could directly assess the impact of NSDHL deficiency on sterol metabolite profiles at cellular resolution.

• CRISPR-based lineage tracing: This approach could track the developmental fate of NSDHL-deficient cells in mosaic tissues, extending observations from mouse models that NSDHL-negative cells are subject to negative selection over time .

These advanced techniques could resolve contradictions in bulk tissue analyses and reveal cell type-specific roles of NSDHL in development and disease.

What computational approaches can integrate structural data with pharmacological screening to develop next-generation NSDHL inhibitors?

Developing next-generation NSDHL inhibitors requires sophisticated computational methods that integrate structural and pharmacological data:

• Structure-based virtual screening: Building on established methodologies that have already identified promising NSDHL inhibitors , future approaches could incorporate molecular dynamics simulations to account for protein flexibility and induced-fit effects during ligand binding.

• Pharmacophore modeling: Developing three-dimensional pharmacophore models based on known NSDHL inhibitors and the enzyme's active site characteristics to guide rational design of improved compounds.

• Quantum mechanics/molecular mechanics (QM/MM) calculations: For detailed investigation of the NSDHL catalytic mechanism and inhibitor interactions at the atomic level.

• Machine learning approaches: Training predictive models on existing structure-activity relationship data to prioritize novel compounds for experimental testing.

• Network pharmacology: Integrating NSDHL inhibition data with broader pathway information (e.g., EGFR signaling networks) to predict system-level effects and potential combination therapies.

• In silico ADMET prediction: Computational prediction of absorption, distribution, metabolism, excretion, and toxicity properties to prioritize compounds with favorable drug-like characteristics early in the development process.

These computational approaches, validated through rigorous experimental testing, could accelerate development of potent and selective NSDHL inhibitors for both research tools and potential therapeutic applications.

How can multi-omics approaches resolve contradictions in our understanding of NSDHL's role in development and disease?

Multi-omics integration offers powerful strategies to address contradictions and knowledge gaps regarding NSDHL function:

• Integrated genomics, transcriptomics, and proteomics: Comparing NSDHL mutation effects across these levels can reveal compensatory mechanisms and explain phenotypic variability. For example, microarray analysis of gene expression in NSDHL-deficient cells has already provided insights into downstream effects of NSDHL mutations .

• Metabolomics profiles: Comprehensive analysis of sterol metabolites and other lipids in NSDHL-deficient versus wild-type cells can clarify which metabolic changes are primary drivers of pathology versus secondary consequences.

• Epigenomic analysis: Investigating whether sterol metabolite accumulation in NSDHL deficiency affects epigenetic regulation, potentially explaining long-term developmental consequences and tissue-specific effects.

• Temporal multi-omics: Analyzing changes across multiple biological levels throughout development could resolve apparent contradictions that may reflect different developmental timepoints.

• Tissue-specific multi-omics: Given the distinct expression patterns of NSDHL across tissues , comprehensive analysis of molecular changes in specific tissues (e.g., brain, skin, liver) could explain tissue-specific manifestations of NSDHL deficiency.

• Systems biology modeling: Integrating multi-omics data into computational models of cholesterol metabolism and signaling networks can simulate the system-wide impact of NSDHL perturbations and generate testable hypotheses about mechanism.

This integrative approach acknowledges the complexity of cholesterol metabolism and its developmental roles, potentially reconciling apparently contradictory findings from different experimental systems.