Recombinant Human Adenosine monophosphate-protein transferase FICD (FICD)

What is FICD and what is its primary function in human cells?

FICD (Filamentation Induced by Cyclic-AMP) is an enzyme responsible for AMPylation, a post-translational modification in which an adenosine monophosphate (AMP) moiety is transferred to target proteins. In human cells, FICD has been exclusively studied as the primary mediator of protein AMPylation . FICD possesses dual catalytic activity - it can both AMPylate and deAMPylate proteins, particularly BiP/HSPA5, a major chaperone in the endoplasmic reticulum (ER) that regulates the unfolded protein response (UPR) . This reversible modification serves as a regulatory mechanism for protein activity, especially during ER stress conditions. FICD's function is particularly critical in differentiated tissues where it helps maintain protein homeostasis over extended periods .

How does FICD-mediated AMPylation regulate cellular stress responses?

FICD regulates cellular stress responses through a sophisticated mechanism involving BiP (HSPA5) AMPylation. During low ER stress or in resting cells, FICD AMPylates BiP, creating an inactive pool of this chaperone in the ER lumen . When ER stress increases, BiP undergoes deAMPylation (also catalyzed by FICD) and returns to an active state to manage the elevated stress levels . This reversible modification serves as a rapid response system that doesn't require new protein synthesis.

The significance of this regulation has been demonstrated across multiple model organisms including Drosophila, Caenorhabditis elegans, rodents, and humans, indicating high evolutionary conservation . Recent studies reveal that FICD is particularly important in mitigating the deleterious effects of UPR activation in tissues associated with UPR-related diseases . This adaptive mechanism appears most crucial in terminally differentiated cells that must maintain long-term resilience to fluctuating environmental and physiological stresses.

What are the known substrates of FICD beyond BiP/HSPA5?

While BiP/HSPA5 is the most well-characterized substrate of FICD, research using chemical-proteomic approaches has identified a diverse range of additional AMPylated proteins. A study utilizing a cell-permeable propargyl adenosine pronucleotide probe identified a total of 162 AMP-modified proteins across various cell types . In HeLa cells specifically, 19 distinct proteins showed AMPylation, including the confirmed substrate HSPA5 as well as novel targets such as PFKP and PPME1 .

FICD dependency studies comparing AMPylation levels in cells with FICD knockdown versus wild-type FICD overexpression (OX) or activated FICD E234G overexpression revealed differential regulation patterns. While HSPA5 showed clear FICD-dependent responses (upregulated AMPylation with FICD E234G OX and downregulated with wild-type FICD OX), other proteins exhibited varying degrees of FICD dependency . Interestingly, MS-based pulldown experiments using chemical crosslinkers confirmed direct interactions between FICD and HSPA5, while proteins like SQSTM1, PFKP, and PPME1 were not found to directly interact with FICD despite being AMPylated . This suggests potential indirect mechanisms or the involvement of additional AMP transferases.

What are the most effective methods for detecting and quantifying FICD-mediated AMPylation in cell systems?

Multiple complementary approaches can be employed for detecting and quantifying FICD-mediated AMPylation, each with specific advantages:

• Chemical-Proteomic Approaches: Cell-permeable propargyl adenosine pronucleotide probes can infiltrate cellular AMPylation pathways, allowing for subsequent detection via click chemistry with fluorescent tags (like rhodamine-azide) . This method has successfully identified AMPylated proteins in intact cancer cell lines, stem cells, neural progenitor cells, neurons, and cerebral organoids.

• Immunoprecipitation with Click Chemistry: After treating cells with an AMPylation probe, specific proteins can be immunoprecipitated and then subjected to click reaction with rhodamine-azide tags to confirm probe incorporation .

• LC-MS/MS Analysis: For comprehensive identification of AMPylated proteins, liquid chromatography-tandem mass spectrometry provides detailed proteome-wide analysis of modified proteins .

• Fluorescence Polarization Assays: For in vitro studies, fluorescent ATP analogs like N6-(6-aminohexyl)-ATP-5-FAM can be used to measure auto-AMPylation of FICD variants. This approach has been successful in high-throughput screening of potential FICD inhibitors .

• Immunohistochemistry Combined with Click Chemistry: This method enables visualization of the cellular localization of both FICD and AMPylated proteins, providing valuable spatial information about AMPylation patterns in different cell types .

For optimal results, researchers should consider combining multiple methods based on their specific experimental questions.

How can researchers effectively manipulate FICD activity for functional studies?

Researchers can manipulate FICD activity through several established approaches:

• Genetic Manipulation:

  • CRISPR-Cas9 Technology: Generation of floxed alleles (like Fic^fl) with LoxP sites positioned to enable Cre-mediated recombination and subsequent gene inactivation .

  • Knockdown Studies: Using RNA interference to reduce FICD expression .

  • Overexpression Systems: Both wild-type FICD and mutant variants can be overexpressed to study gain-of-function effects .

• Mutant Variants:

  • FICD E234G Mutant: This mutant acts as a deAMPylation-deficient, constitutive AMPylase due to conformational changes in the FICD inhibitory helix that relieve autoinhibition . It's particularly useful for studying AMPylation activity in isolation.

  • Catalytically Inactive Mutants: Can serve as negative controls in functional studies.

• Chemical Inhibitors:

  • Small-molecule FICD inhibitors have been identified using fluorescence polarization screening assays . These compounds can provide temporal control over FICD activity without the need for genetic manipulation.

• Experimental Parameters:

  • Optimal buffer conditions for in vitro FICD activity include 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, with typical protein concentrations of 1 μM FICD and 0.25 μM ATP analog .

When designing functional studies, it's important to consider the dual AMPylation/deAMPylation activity of FICD and how experimental manipulations might differentially affect these functions.

What experimental systems are most suitable for studying FICD function in neurogenesis?

Based on the research findings, the following experimental systems have proven valuable for studying FICD's role in neurogenesis:

• Cerebral Organoids (COs): These three-dimensional culture systems have successfully demonstrated FICD-dependent AMPylation remodeling and its effect on accelerating differentiation of neural progenitor cells into mature neurons . COs provide a complex, physiologically relevant environment that recapitulates aspects of human brain development.

• Neural Progenitor Cells (NPCs): These cells show distinct AMPylation patterns, with modifications strictly localized next to the rough ER and in the nucleus . NPCs serve as an excellent model for studying the transition from progenitor to differentiated neuron states.

• Primary Neurons: In differentiated neurons, AMPylation has been observed in the nucleus and to a lesser extent in neurites, including MAP2+ dendrites and phospho-TAU+ axons . This system allows for detailed study of AMPylation in post-mitotic neural cells.

• Cell Lines with Neural Potential: Human-derived stem cells can be directed toward neural lineages to study how FICD activity changes during differentiation processes .

• Combined Imaging Approaches: Techniques combining click chemistry with rhodamine-azide for intracellular probe visualization and immunohistochemistry for FICD and various cellular markers (PDI for ER, GM130 for Golgi complex, TUBB3 for neuronal microtubule cytoskeleton, MAP2 for dendrites, phospho-TAU for axons) have been particularly informative in revealing the cellular and subcellular distribution of AMPylation in neural cells .

When selecting an experimental system, researchers should consider that the localization patterns of FICD and AMPylated proteins differ significantly across cell types, suggesting potentially distinct regulatory mechanisms.

How does the dual AMPylation/deAMPylation activity of FICD contribute to cellular homeostasis?

FICD's dual catalytic activity represents a sophisticated regulatory mechanism for maintaining cellular homeostasis, particularly during stress conditions. This bifunctional enzyme can both transfer AMP to proteins (AMPylation) and remove AMP from proteins (deAMPylation), with the balance between these activities shifting in response to cellular stress levels .

During normal conditions or low ER stress, FICD predominantly functions as an AMPylase, creating a reserve pool of inactive BiP/HSPA5 in the ER lumen . When stress increases, FICD's activity shifts toward deAMPylation, rapidly mobilizing the BiP reserve without requiring new protein synthesis . This rapid response capability is crucial for managing acute stress situations.

Studies across multiple model organisms (Drosophila, C. elegans, rodents, and humans) have confirmed the conservation of this mechanism, underscoring its fundamental importance . The regulatory significance appears most pronounced in terminally differentiated cells that must maintain protein homeostasis over extended periods rather than in cells with high regenerative capacity .

Recent research has expanded our understanding of how FICD's dual activity is regulated at the molecular level. Conformational changes in the FICD inhibitory helix appear to control the balance between AMPylation and deAMPylation activities . The E234G mutation, which disrupts this regulatory mechanism, results in constitutive AMPylation activity without deAMPylation capability .

What is the relationship between FICD activity and the unfolded protein response in disease contexts?

FICD plays a critical role in tempering the unfolded protein response (UPR), particularly in terminally differentiated tissues where cellular resilience to stress is essential for preventing disease . As differentiated cells must maintain function throughout an organism's lifespan, the regulation of protein homeostasis becomes crucial for preventing conditions associated with protein misfolding and ER stress.

The relationship between FICD and disease contexts appears to be tissue-specific and sensitive to the nature of the stress. Research indicates that FICD is required for the regulation of UPR during both physiological and pharmacological stresses . In tissues with limited regenerative capacity, FICD-mediated AMPylation provides a critical mechanism for preventing excessive or prolonged UPR activation, which can lead to cell death and tissue dysfunction.

Notably, researchers have predicted that FICD is important in mitigating deleterious effects of UPR activation in various tissues with UPR-associated diseases, positioning FICD as a promising therapeutic target . The specific disease contexts could include neurodegenerative disorders, diabetes, and other conditions characterized by ER stress and protein misfolding.

A particularly interesting observation is that cells with high regenerative capacity may not require this level of UPR regulation, as new cells will bypass the need for long-term survival . This suggests that FICD-targeted therapeutic approaches might need to be tailored to specific tissues and disease contexts.

How do the subcellular localization patterns of FICD and AMPylated proteins contribute to their function?

The subcellular localization of FICD and AMPylated proteins varies significantly across cell types and appears to be functionally relevant. Detailed imaging studies combining click chemistry with immunohistochemistry have revealed distinct localization patterns :

• In HeLa cells: AMPylated proteins are enriched in the nucleus, with additional small spots in the cytoplasm partially overlapping with the ER. FICD itself is localized in the ER .

• In Neural Progenitor Cells (NPCs): AMPylation is strictly localized next to the rough ER and in the nucleus .

• In Neurons: AMPylation is observed in the nucleus and to a lesser extent in neurites, including both dendrites (MAP2+) and axons (phospho-TAU+) .

• In Fibroblasts: AMPylation accumulates around the nucleus but is completely absent inside it .

These distinct localization patterns suggest cell type-specific functions for AMPylation. The observation that FICD and AMPylated proteins don't always colocalize supports the hypothesis that additional AMP transferases with complementary cellular distribution may exist .

The ER localization of FICD has been confirmed through multiple approaches, including C-terminal FLAG tag experiments and endoglycosidase H assays . This localization is consistent with FICD's role in regulating BiP/HSPA5, an ER chaperone, and more broadly with its function in the unfolded protein response.

The nuclear localization of AMPylated proteins across multiple cell types points to potential roles for this modification in regulating nuclear processes, which remains an area for further investigation.

What are the major challenges in studying FICD-mediated AMPylation in live cells?

Studying FICD-mediated AMPylation in live cells presents several technical challenges that researchers must overcome:

• Probe Delivery Limitations: ATP-derived probes traditionally used for studying AMPylation suffer from restricted cellular uptake due to their charged nature . Additionally, these probes face competition with high endogenous ATP levels, potentially reducing sensitivity.

• Specificity Concerns: Distinguishing FICD-mediated AMPylation from modifications by other potential AMP transferases requires careful experimental design. The discovery that localization patterns of FICD and AMPylated proteins don't always overlap suggests the presence of additional, currently unidentified AMP transferases .

• Dynamic Nature of Modification: The dual AMPylation/deAMPylation activity of FICD creates a dynamic equilibrium that can be difficult to capture at specific time points .

• Cell Type Variability: The significant differences in AMPylation patterns across cell types necessitates optimization of experimental approaches for each system .

To address these challenges, researchers have developed innovative solutions:

• Cell-Permeable Pronucleotide Probes: These probes can infiltrate cellular AMPylation pathways more effectively than traditional ATP derivatives .

• Genetic Manipulation Approaches: CRISPR-Cas9 technology for generating conditional knockout models and overexpression systems for wild-type and mutant FICD variants provide valuable tools for dissecting FICD-specific effects .

• Combining Multiple Detection Methods: Using complementary approaches such as chemical proteomics, immunoprecipitation, click chemistry, and imaging provides a more comprehensive view of AMPylation dynamics .

• Enzyme Mutation Studies: Utilizing FICD variants like E234G that selectively affect either AMPylation or deAMPylation helps isolate specific aspects of FICD function .

How can researchers accurately quantify and compare the efficacy of FICD inhibitors?

Accurate quantification and comparison of FICD inhibitor efficacy requires robust methodological approaches:

• Fluorescence Polarization (FP) Assays: These in vitro assays can measure auto-AMPylation of FICD E234G using fluorescent ATP analogs like N6-(6-aminohexyl)-ATP-5-FAM . Key parameters for optimal results include:

  • Enzyme concentration: 1 μM FICD

  • Substrate concentration: 0.25 μM FL-ATP

  • Buffer composition: 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT

  • Temperature: 37°C

  • Measurement: Fluorescence polarization readings using 485/530 nm filters

• Fractional Inhibitory Concentration (FIC) Index Analysis: This approach can be adapted from antimicrobial research to assess FICD inhibitor efficacy . For each inhibitor, the FIC is calculated as:
  FIC = (Concentration of inhibitor in combination) / (MIC of inhibitor alone)

  For combinations of inhibitors, the sum of FICs (ΣFIC) provides insight into potential synergistic or antagonistic effects:
  ΣFIC = FICA + FICB = (CA/MICA) + (CB/MICB)

• Statistical Validation: Monte Carlo simulation analysis can strengthen the reliability of inhibitor comparison:

  • Transform FICs to log2 values to approximate normal distribution

  • Calculate average and standard deviation

  • Perform normality testing (e.g., D'Agostino-Pearson)

  • Use mean and SD in Monte Carlo analysis to simulate multiple experiments

  • Assess interactions based on whether the 95% confidence interval includes a predefined cutoff

• Cellular Validation Assays: Beyond in vitro testing, inhibitor efficacy should be evaluated in cellular contexts by measuring:

  • Changes in AMPylation levels of known FICD substrates (e.g., BiP/HSPA5)

  • Functional impact on the unfolded protein response

  • Potential off-target effects on related cellular processes

By combining these approaches, researchers can develop a comprehensive profile of inhibitor efficacy and specificity.

What methodological considerations are important when studying FICD in different cellular contexts?

When studying FICD across different cellular contexts, researchers should consider several methodological adaptations:

• Cell Type-Specific Localization Patterns: Research has shown that both FICD and AMPylated proteins display distinct localization patterns across cell types . Methods must be optimized to capture these differences:

  • In HeLa cells: Focus on nuclear and ER-associated AMPylation

  • In NPCs: Examine regions next to rough ER and nucleus

  • In neurons: Include analysis of both nuclear and neurite (dendrite/axon) regions

  • In fibroblasts: Pay particular attention to perinuclear regions

• Differentiation Stage Considerations: FICD function appears to vary with cellular differentiation state :

  • In stem cells and progenitors: Consider the role of FICD in differentiation processes

  • In terminally differentiated cells: Focus on long-term stress resilience functions

• Marker Selection for Colocalization Studies: Different cellular markers provide context-specific information when combined with AMPylation detection:

  • PDI for rough endoplasmic reticulum

  • GM130 for Golgi complex

  • TUBB3 for total neuronal microtubule cytoskeleton

  • MAP2 for neuronal dendrites

  • Phospho-TAU for neuronal axons

  • DAPI for nuclei visualization

• Stress Condition Relevance: The dual AMPylation/deAMPylation activity of FICD responds differently to various stress conditions:

  • Physiological versus pharmacological stressors may elicit different responses

  • The duration and intensity of stress can affect the balance of AMPylation/deAMPylation

• Model System Selection: Different model systems offer complementary insights:

  • Cell lines provide controlled conditions for mechanistic studies

  • Primary cells reflect more physiological contexts

  • Cerebral organoids capture complex cellular interactions and developmental processes

  • Animal models (with appropriate FICD genetic modifications) enable tissue-specific and systemic analysis

• Temporal Considerations: The dynamic nature of FICD-mediated AMPylation requires appropriate timepoint selection:

  • Early timepoints may capture initial stress responses

  • Later timepoints reflect adaptation and homeostatic adjustments

What are the potential therapeutic applications of targeting FICD in diseases associated with ER stress?

FICD represents a promising therapeutic target for diseases associated with ER stress and dysregulated unfolded protein response (UPR) pathways. Research indicates that FICD is required for regulating the UPR during both physiological and pharmacological stresses, particularly in differentiated tissues with limited regenerative capacity .

Potential therapeutic applications include:

• Neurodegenerative Disorders: Given FICD's role in neurogenesis and neuronal differentiation, modulating its activity could potentially address the ER stress component of conditions like Alzheimer's, Parkinson's, and ALS . The finding that FICD-dependent AMPylation remodeling accelerates differentiation of neural progenitor cells into mature neurons suggests potential applications in neural regeneration strategies.

• Metabolic Diseases: The involvement of FICD in regulating BiP/HSPA5, a key component of the UPR, suggests potential applications in diseases like diabetes where ER stress plays a significant role . GO term analysis of FICD interacting partners has indicated a link to basal metabolism, further supporting this direction .

• Cancer Therapy: The differential expression and activity of FICD across cell types, particularly between highly proliferative and differentiated cells, suggests potential for cancer-specific targeting strategies .

• Protection of Post-Mitotic Tissues: FICD's importance in maintaining long-term cellular resilience in differentiated tissues presents opportunities for developing protective therapies for tissues with limited regenerative capacity .

Development of small-molecule FICD inhibitors is already underway, with fluorescence polarization assays being used to identify compounds that suppress both endogenous and auto-AMPylation activities . The bifunctional nature of FICD (both AMPylation and deAMPylation) presents opportunities for developing drugs that selectively modulate specific aspects of its activity based on disease context.

How might FICD function interact with other post-translational modification systems in complex cellular networks?

FICD-mediated AMPylation likely operates within a complex network of post-translational modifications (PTMs) that collectively regulate cellular homeostasis. Understanding these interactions presents significant opportunities for advanced research:

• Integration with Phosphorylation Networks: As both AMPylation and phosphorylation involve the transfer of groups from ATP, there may be regulatory crosstalk between these systems. This is particularly relevant given that pseudokinases (which account for approximately 10% of the human kinome) have been found to possess AMP transferase activity .

• UPR-Associated Modification Cascades: Within the unfolded protein response pathway, multiple PTMs including ubiquitination, SUMOylation, and acetylation work alongside AMPylation to regulate protein function. Research into how these systems interact could reveal hierarchical or cooperative regulation patterns.

• Compartmentalization of PTM Systems: The distinct localization patterns of AMPylated proteins across cell types suggests potential compartment-specific interactions with other PTM systems. Nuclear AMPylation, for example, may interact with histone modifications to influence gene expression.

• Temporal Dynamics Between PTM Systems: Different PTM systems operate on various timescales. AMPylation's role in creating a rapidly mobilizable reserve of inactive BiP may be complemented by slower but more sustained regulatory mechanisms.

• Stress-Specific PTM Interactions: Different stress conditions might favor specific interactions between AMPylation and other PTM systems. For example, oxidative stress, which affects many PTMs through redox-sensitive mechanisms, could create unique interaction patterns with the AMPylation system.

• Computational Modeling Opportunities: The complexity of these interactions presents an opportunity for systems biology approaches to model PTM crosstalk networks and predict emergent regulatory behaviors.

Future research utilizing multi-omic approaches that simultaneously track multiple PTMs could provide critical insights into these complex regulatory networks.

What is the evolutionary significance of FICD-mediated AMPylation across different species?

The high conservation of FICD and its AMPylation mechanisms across diverse species highlights its fundamental importance in cellular homeostasis. Research has confirmed similar functions across Drosophila, Caenorhabditis elegans, rodents, and humans , suggesting strong evolutionary pressure to maintain this regulatory system.

Several aspects of FICD's evolutionary significance merit further investigation:

• Adaptation to Cellular Complexity: The increasing importance of FICD in differentiated, long-lived cells versus those with high regenerative capacity suggests that this system may have evolved to support the emergence of complex multicellular organisms with specialized, terminally differentiated tissues.

• Conservation of Substrate Specificity: While BiP/HSPA5 is a conserved FICD substrate across species, the full range of AMPylated proteins may vary, potentially reflecting species-specific adaptations to different physiological demands or environmental stresses.

• Co-evolution with the UPR System: The integration of FICD into the unfolded protein response pathway suggests co-evolutionary development of these systems. Comparative genomic studies across evolutionary distant species could reveal how this relationship developed.

• Stress Response Adaptation: Different species face varying environmental stresses, and FICD-mediated AMPylation may have adapted to specific stress patterns encountered during a species' evolutionary history.

• Dual Catalytic Activity Development: The evolution of both AMPylation and deAMPylation activities within a single enzyme represents an elegant regulatory solution. Understanding how this dual functionality emerged could provide insights into enzyme evolution more broadly.

• Relationship to Other AMP Transferases: The recent discovery that selenoprotein-O (SelO) and potentially other pseudokinases possess AMP transferase activity raises questions about the evolutionary relationships between different AMPylation systems.

These evolutionary considerations not only provide theoretical insights but also have practical implications for using model organisms in FICD research and for developing therapeutic approaches based on this highly conserved system.

Optimal Conditions for FICD Activity Assays

Cellular Localization Patterns of FICD and AMPylated Proteins

These tables synthesize key findings from the available research on FICD, providing a foundation for researchers to design experiments, interpret results, and develop new hypotheses in this rapidly evolving field.