Recombinant Pongo abelii Squalene synthase (FDFT1)

What is FDFT1 and what is its primary function in cellular metabolism?

FDFT1 (Farnesyl-diphosphate farnesyltransferase 1), also known as squalene synthase (SQS), is a 416-amino acid, 47-kDa membrane-associated enzyme located almost exclusively in the endoplasmic reticulum. Its primary function is to synthesize squalene via condensation of two molecules of farnesyl pyrophosphate (FPP). This represents a critical step in the cholesterol biosynthesis pathway, as FDFT1 catalyzes the first committed step toward sterol synthesis .

The enzyme functions through a two-stage reaction process: first, dimerization of two FPP molecules forms pre-squalene diphosphate (PSDP), which is then rearranged and reduced by NADPH to create squalene. This reaction requires a divalent cation, preferably Mg²⁺, to bind to and extract a diphosphate group, similar to other prenyltransferases .

How conserved is FDFT1 across species and what does this suggest about its evolutionary importance?

The high conservation of FDFT1 across diverse species indicates its fundamental importance in cellular metabolism. The human FDFT1 protein sequence shares between 86% and 93% identity with other mammals, suggesting strong evolutionary pressure to maintain its function. Beyond mammals, FDFT1 is present in yeast, bacteria, and plants, further emphasizing its critical importance throughout evolution .

The Pongo abelii (Sumatran orangutan) FDFT1 exhibits high homology with human FDFT1, making it a valuable model for studying the enzyme's function in contexts relevant to human health and disease. This conservation across species enables comparative studies that can yield insights into the fundamental mechanisms of sterol biosynthesis .

What are the key domains and active sites of FDFT1 that are critical for its enzymatic function?

The catalytic mechanism of FDFT1 involves multiple conserved domains that facilitate substrate binding and catalysis. While the search results don't provide complete details on all domains, we know that FDFT1 requires specific binding sites for FPP, NADPH, and divalent cations (particularly Mg²⁺). The enzyme possesses active sites involved in both the dimerization of FPP to form PSDP and the subsequent rearrangement and reduction steps .

Understanding these domains is essential for structure-based drug design targeting FDFT1. For experimental approaches to study these domains, researchers typically employ site-directed mutagenesis of conserved residues followed by activity assays to determine their contribution to catalysis or substrate binding.

What are the optimal conditions for expressing and purifying recombinant Pongo abelii FDFT1 for structural and functional studies?

For optimal expression and purification of recombinant Pongo abelii FDFT1, researchers should consider the following approach:

• Expression system selection: E. coli systems work for partial protein domains, but mammalian or insect cell systems are recommended for full-length FDFT1 due to its membrane association and potential post-translational modifications.

• Tag selection: Based on available data, the tag type should be optimized during the production process to ensure proper folding and function .

• Buffer optimization: Use Tris-based buffers with 50% glycerol for storage, as indicated for commercially available recombinant protein preparations .

• Storage conditions: Store at -20°C for regular use, or -80°C for extended storage. Working aliquots can be maintained at 4°C for up to one week. Repeated freeze-thaw cycles should be avoided .

The specific expression region (amino acids 1-417) should be considered when designing constructs, as this represents the full-length protein in Pongo abelii FDFT1 .

What methodological approaches can be used to study FDFT1's interaction with its binding partners in vitro?

To study FDFT1's interactions with binding partners, researchers can employ several complementary techniques:

• Affinity-based approaches: Pull-down assays using tagged FDFT1 can identify interacting proteins, as previous research has demonstrated 72 experimentally proven interacting partners according to GeneCards and String databases .

• Yeast two-hybrid screening: This approach has successfully identified FDFT1 binding partners in previous studies .

• Co-immunoprecipitation: For confirming physiologically relevant interactions in cellular contexts.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): To determine binding kinetics and thermodynamic parameters.

• Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET): For studying real-time interactions in living cells.

When designing such experiments, researchers should consider that FDFT1 is membrane-associated, which may complicate some interaction studies and necessitate the use of appropriate detergents or membrane-mimetic systems.

How can researchers effectively measure FDFT1 enzymatic activity in different experimental systems?

Measuring FDFT1 enzymatic activity can be approached through several methodologies:

• Radiometric assays: Using radiolabeled FPP as substrate and measuring the formation of radiolabeled squalene.

• HPLC or LC-MS based assays: For quantifying the conversion of FPP to squalene and/or the formation of PSDP as an intermediate.

• Coupled enzyme assays: Measuring NADPH consumption, as FDFT1 requires NADPH for the reduction step of its reaction.

• In vivo metabolic labeling: Using isotope-labeled substrates to track flux through the pathway.

For all these approaches, researchers must consider:

• The requirement for Mg²⁺ or other divalent cations in the reaction buffer

• The need for detergents or membrane fractions when working with the membrane-bound enzyme

• The potential influence of other cellular factors on activity measurements

How does FDFT1 contribute to cancer progression, and what are the mechanisms underlying its role in metabolic reprogramming?

FDFT1 contributes to cancer progression through several interrelated mechanisms:

• Metabolic reprogramming: As a key enzyme in cholesterol biosynthesis, FDFT1 supports the increased demand for membrane components in rapidly proliferating cancer cells. This metabolic adaptation is critical for maintaining cellular structures during rapid division .

• Cell proliferation regulation: Inhibition of FDFT1 expression significantly attenuates cell proliferation, increases sub-G1, G1, and G2/M-phase populations, and decreases S-phase population, suggesting its importance in cell cycle progression .

• Anti-apoptotic signaling: FDFT1 can directly activate NF-κB pathways or activate AKT through cholesterol synthesis. Activated NF-κB increases anti-apoptotic proteins such as Bcl-xL, Bcl-2, and Bax while decreasing pro-apoptotic proteins like caspase-3, thereby blocking apoptosis signaling .

• Protection against ferroptosis: In cancer cells with deficient squalene epoxidase, high expression of FDFT1 increases intracellular squalene levels, which protects the cell membrane from lipid peroxidation by reactive oxygen species (ROS) and prevents ferroptosis .

• Autophagy regulation: FDFT1 inhibition upregulates endogenous geranylgeranoic acid (GGA) content, resulting in incomplete autophagy, while reduction in cholesterol levels induces autophagy .

These mechanisms collectively support cancer cell survival and proliferation, making FDFT1 a potential target for cancer therapy.

What is the clinical relevance of FDFT1 as a prognostic marker in different cancer types?

FDFT1 has emerging significance as a prognostic marker in various cancer types, although the search results don't provide comprehensive details on specific cancer types. The available evidence indicates that:

• FDFT1 could serve as a potential cancer prognostic marker based on its critical role in metabolic reprogramming, cell proliferation, and invasion .

• Its contribution to the hallmarks of cancer, particularly through metabolic pathways, suggests its utility in predicting cancer progression and potentially treatment response .

• The enzyme's role in directing metabolic flux toward either sterol or non-sterol pathways may be particularly relevant in cancers that demonstrate altered lipid metabolism.

For researchers exploring FDFT1 as a prognostic marker, a comprehensive approach would include:

• Correlation of FDFT1 expression with survival outcomes in patient cohorts

• Multivariate analysis controlling for established prognostic factors

• Evaluation of FDFT1 activity (not just expression) as a potential biomarker

• Integration with other metabolic markers for improved prognostic value

How does FDFT1 regulate the metabolic flux between sterol and non-sterol branches, and what cellular factors influence this regulation?

FDFT1 serves as a critical branch-point enzyme that determines whether farnesyl pyrophosphate (FPP) is directed toward sterol synthesis or non-sterol pathways. This regulatory function involves several mechanisms:

• Substrate competition: FDFT1 competes with other enzymes that use FPP as a substrate, including those involved in protein prenylation, dolichol synthesis, and ubiquinone production. The relative activities of these enzymes help determine metabolic flux .

• Enzymatic regulation: The regulation of FDFT1 activity is thought to play an important role in determining whether FPP is switched toward either sterol or nonsterol branches in response to changing cellular requirements .

• Feedback mechanisms: Products of the cholesterol pathway, particularly oxysterols, can regulate FDFT1 through transcriptional mechanisms involving SREBP processing .

• Cellular factors influencing regulation:

  • Cellular cholesterol levels

  • Cellular energy status (NADPH availability)

  • Presence of divalent cations (particularly Mg²⁺)

  • Activity of upstream enzymes in the mevalonate pathway

  • Intracellular signaling pathways that modulate FDFT1 activity

Understanding this regulatory node is particularly important because non-sterol products derived from FPP include essential compounds like ubiquinone and heme A (for the mitochondrial electron transport chain), dolichol (for glycoprotein synthesis), and prenylated proteins (including G-proteins, Ras, and p21) that are crucial for cell signaling and growth regulation .

What are the transcriptional and post-translational mechanisms that regulate FDFT1 expression and activity?

The regulation of FDFT1 occurs through multiple mechanisms at both transcriptional and post-translational levels:

Transcriptional Regulation:

• SREBP pathway: Sterol Regulatory Element-Binding Proteins (SREBPs) are key transcription factors that regulate FDFT1 expression in response to cellular sterol levels.

• Tissue-specific expression: FDFT1 is expressed at particularly high levels in the hypothalamus and liver, suggesting tissue-specific transcriptional regulation .

• Pathogen-responsive regulation: In plants, FDFT1 expression shows host-specific transcriptional regulation during pathogen infection. For example, NbSQS (the plant homolog) was significantly induced at 48 hours post-inoculation with P. infestans, while SlSQS expression was significantly suppressed at 12 and 24 hours post-inoculation .

Post-translational Regulation:

• Protein-protein interactions: The 72 experimentally proven interacting proteins may play roles in regulating FDFT1 activity through direct interactions .

• Membrane localization: As a membrane-associated enzyme found almost exclusively in the endoplasmic reticulum, FDFT1's subcellular localization may be regulated to control its activity .

• Metabolite regulation: Intermediates in the cholesterol biosynthesis pathway, including oxysterols and farnesyl pyrophosphate, may affect FDFT1 activity through allosteric mechanisms.

Understanding these regulatory mechanisms provides potential targets for modulating FDFT1 activity in research and therapeutic contexts.

How has the function of FDFT1 been adapted across different organisms, and what can this tell us about its evolutionary significance?

The evolutionary conservation of FDFT1 across diverse species—from bacteria and yeast to plants and mammals—highlights its fundamental importance in cellular metabolism. Several key observations about its evolutionary adaptation include:

• Core function conservation: The basic catalytic function of converting FPP to squalene is preserved across diverse organisms, indicating the fundamental importance of this metabolic step.

• Differential regulation: While the catalytic function is conserved, regulatory mechanisms vary across species. For example, plant SQS (the FDFT1 homolog) shows host-specific transcriptional regulation during pathogen infection, with different expression patterns in different plant species .

• Role in different physiological contexts:

  • In mammals: Primarily functions in cholesterol biosynthesis, with implications for membrane structure, hormone production, and bile acid synthesis.

  • In plants: Contributes to phytosterol production and plays roles in plant-pathogen interactions, with overexpression in N. benthamiana reducing resistance against P. infestans .

  • In microorganisms: Often involved in the production of ergosterol or other membrane sterols.

• Metabolic integration: The integration of FDFT1 within larger metabolic networks shows species-specific adaptations while maintaining its core function.

This evolutionary perspective provides important insights for researchers:

• Conserved domains likely represent functionally critical regions

• Species-specific differences may highlight adaptive specializations

• Comparative studies can reveal fundamental aspects of sterol metabolism

What are the most effective analytical techniques for studying FDFT1 expression and activity in different experimental systems?

Researchers have multiple analytical options for studying FDFT1, each with specific applications:

For FDFT1 Expression Analysis:

• Quantitative RT-PCR: Provides specific measurement of FDFT1 mRNA levels, as demonstrated in studies measuring NbSQS expression upon P. infestans infection .

• Western blotting: For protein-level detection and semi-quantitative analysis.

• RNA-Seq: For transcriptome-wide analysis of FDFT1 expression in relation to other genes, as shown in comparative transcriptomics of wild type and FDFT1-overexpressing plants .

• Immunohistochemistry: For spatial localization within tissues.

For FDFT1 Activity Measurement:

• Metabolite quantification: Measuring downstream products like squalene or phytosterols using:

  • Gas chromatography-mass spectrometry (GC-MS)

  • Liquid chromatography-mass spectrometry (LC-MS)

  • Nuclear magnetic resonance (NMR) spectroscopy

• Enzyme assays: Measuring the conversion of FPP to squalene in vitro.

• Metabolic flux analysis: Using isotope-labeled substrates to track carbon flow through the pathway.

Considerations for Experimental Design:

• Temporal dynamics: Expression can change significantly over time, as demonstrated by the time-course analyses in plant pathogen studies (e.g., significant induction at 48 hpi but not earlier) .

• Tissue specificity: Expression levels vary by tissue, with particularly high levels in hypothalamus and liver in humans .

• Species differences: Experimental approaches may need adjustment based on species-specific characteristics of FDFT1, particularly when comparing across evolutionary distant organisms.

What are the key considerations for designing inhibition studies targeting FDFT1, and how can researchers overcome common challenges?

When designing inhibition studies targeting FDFT1, researchers should consider several critical factors:

Key Considerations:

• Selectivity: FDFT1 inhibitors should be selective against other enzymes in the mevalonate pathway to avoid confounding effects. FDFT1's position downstream of HMG-CoA reductase means that selective inhibition does not affect the synthesis of non-sterol isoprenoids like dolichol, ubiquinone, heme A, and isoprenylated proteins .

• Mechanism-based approaches: Consider the two-stage reaction mechanism (FPP dimerization to PSDP, followed by PSDP reduction to squalene) when designing inhibitors. Different compounds may target different steps.

• Membrane association: FDFT1 is membrane-associated, which can affect inhibitor access and efficacy in cellular contexts.

• Phenotypic readouts: When evaluating inhibitors, consider multiple potential consequences of FDFT1 inhibition:

  • Cell cycle effects (sub G1, G1, G2/M-phase populations)

  • Apoptotic signaling (NF-κB pathways, AKT activation)

  • Autophagy induction

Common Challenges and Solutions:

• Challenge: Membrane permeability of inhibitors
  Solution: Structure-activity relationship studies to optimize physicochemical properties for membrane penetration

• Challenge: Off-target effects
  Solution: Employ multiple structurally distinct inhibitors and genetic approaches (siRNA, CRISPR) to confirm target specificity

• Challenge: Compensatory metabolic mechanisms
  Solution: Combine inhibitor studies with metabolomics to identify potential bypass pathways

• Challenge: Species differences in FDFT1 structure
  Solution: Consider species-specific differences when testing inhibitors across model systems

These considerations are particularly important for researchers exploring FDFT1 as a potential target for cancer treatment, as suggested by its critical role in cancer metabolic reprogramming, cell proliferation, and invasion .

What are the emerging research areas exploring FDFT1's role beyond cholesterol biosynthesis?

Several emerging research areas are exploring FDFT1's functions beyond its canonical role in cholesterol biosynthesis:

• Immune system modulation: Pre-squalene diphosphate (PSDP), an intermediate in the FDFT1 reaction, directly inhibits phospholipase D and leukocyte activities, leading to down-regulation of intracellular signals that decrease the amplitude of acute inflammatory responses. This suggests FDFT1 may have immunomodulatory functions through PSDP production .

• Plant-pathogen interactions: Research in plants has revealed that FDFT1 homologs (SQS) play important roles in plant resistance against pathogens. Overexpression of SQS in N. benthamiana reduced plant resistance against P. infestans and induced hyperaccumulation of stigmasterol, suggesting complex roles in plant immunity .

• Transcriptional network effects: Comparative transcriptomics analysis of SQS-overexpressing plants showed that diverse plant physiological processes were influenced, including sulfur compound biosynthesis, copper ion transport, response to hydrogen peroxide, phenylpropanoid metabolism, and sesquiterpene biosynthesis. This suggests that FDFT1/SQS may have broad regulatory effects beyond direct metabolic functions .

• Genomic stability: FDFT1 interacts with proteins involved in maintaining genomic stability, such as WWOX (WW domain-containing oxidoreductase). WWOX deletion in mouse B cells leads to genome instability, and somatic loss of WWOX is associated with TP53 perturbation in basal-like breast cancer. This potential relationship between FDFT1 and genomic stability warrants further investigation .

• Non-canonical signaling pathways: FDFT1 may influence cellular physiology through interactions with its 72 experimentally proven binding partners, many of which are involved in diverse cellular processes beyond cholesterol metabolism .

These emerging areas represent promising directions for researchers seeking to understand the broader biological significance of FDFT1.

How might comparative studies of FDFT1 across different species contribute to our understanding of fundamental biological processes?

Comparative studies of FDFT1 across species offer valuable insights into fundamental biological processes:

• Evolutionary conservation of metabolic pathways: The high conservation of FDFT1 across diverse organisms highlights the fundamental importance of sterol biosynthesis throughout evolution. Comparing the enzyme across species can reveal which aspects of its structure and function have been most strongly conserved, pointing to critical functional domains .

• Species-specific adaptations: Different organisms use sterols for various purposes, and studying FDFT1 adaptations can reveal how metabolic pathways evolve to meet specific physiological needs. For example, plants utilize phytosterols for both membrane structure and defense against pathogens .

• Differential regulation mechanisms: The contrasting regulation of FDFT1/SQS expression observed between plant species during pathogen infection (induction in N. benthamiana vs. transient suppression in S. lycopersicum) highlights how the same enzyme can be regulated differently across species in response to similar challenges .

• Pathway integration variations: Comparative transcriptomics of FDFT1-overexpressing plants revealed diverse physiological processes affected by FDFT1 modulation, suggesting species-specific integration of sterol metabolism with other cellular pathways .

• Model system validation: Understanding the similarities and differences in FDFT1 structure and function across species helps researchers select appropriate model systems and interpret findings in the context of human health and disease.

Comparative approaches might include:

• Phylogenetic analysis of FDFT1 sequence conservation

• Structural comparisons across species to identify conserved domains

• Cross-species functional complementation studies

• Metabolic profiling across species with FDFT1 modulation

These approaches can provide broader insights into metabolic pathway evolution, host-pathogen interactions, and the fundamental principles of enzyme regulation across the tree of life.