Recombinant Mouse Adenosine monophosphate-protein transferase FICD (Ficd)

What is FICD and what is its primary function in cellular biology?

FICD (Fic domain-containing protein) is a bifunctional enzyme capable of both AMPylation (adding AMP) and deAMPylation (removing AMP) of target proteins. This post-translational modification system plays a critical role in regulating the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. In its native context, FICD regulates adaptation to UPR by modifying BiP, an essential ER chaperone. The enzyme has been shown to AMPylate BiP during homeostasis and deAMPylate it during stress conditions, making it a critical regulator of protein folding dynamics and cellular stress responses .

How does FICD's structure relate to its enzymatic function?

FICD contains a conserved Fic domain with the characteristic Fic motif (typically HPFDDGNGR) that catalyzes the transfer of AMP from ATP to target proteins. The protein also contains an inhibitory helix with a conserved glutamate residue (E to G mutation creates a deregulated, constitutively AMPylation-active enzyme). This structural arrangement is critical for regulating the switch between AMPylation and deAMPylation activities. The enzyme's ability to switch between these functions appears to be regulated by dimerization in some cases, where the dimer interface is linked to the active site. Dimerization can transfer rigidity toward the inhibitory helix, preventing AMPylation and favoring deAMPylation .

How does the activity of FICD differ from AMPK despite both involving AMP?

Despite both involving AMP in their activities, FICD and AMPK (Adenosine Monophosphate-activated Protein Kinase) are distinct enzymes with different functions:

Unlike FICD, AMPK is activated allosterically by AMP and by phosphorylation, serving as a key metabolic regulator that senses the energy status of cells .

What are the optimal conditions for expressing recombinant mouse FICD in bacterial systems?

For optimal expression of enzymatically active recombinant mouse FICD:

• Expression system: E. coli BL21(DE3) strains are recommended due to their reduced protease activity. For proper folding, consider Origami or SHuffle strains that facilitate disulfide bond formation.

• Expression conditions:

  • Temperature: Induce at 18°C for 16-20 hours to enhance proper folding

  • IPTG concentration: 0.1-0.5 mM (optimize for your specific construct)

  • Media: Use enriched media (such as Terrific Broth) supplemented with glucose

• Construct design:

  • Include an N-terminal His-tag or GST-tag for purification

  • Consider including a TEV protease cleavage site for tag removal

  • For functional analysis, prepare both wild-type and E66G (inhibitory helix) mutants

• Purification protocol:

  • Use immobilized metal affinity chromatography followed by size exclusion chromatography

  • Maintain reducing conditions with 1-2 mM DTT or β-mercaptoethanol

  • Preferred buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 1 mM DTT

These conditions should be optimized for each specific construct and experimental requirement.

How can I design experiments to differentiate between AMPylation and deAMPylation activities of mouse FICD?

To differentiate between these opposing enzymatic activities:

• Preparation of enzyme variants:

  • Wild-type FICD: Will show minimal AMPylation in standard conditions due to inhibitory helix regulation

  • E66G mutant: Deregulated variant with enhanced AMPylation activity

  • H205A mutant: Catalytically inactive control (mutation in the Fic motif)

• Detection methods:

  • Western blotting with anti-AMP antibodies to detect AMPylated proteins

  • Mass spectrometry to identify modified residues (e.g., MS/MS has confirmed S10 as a site of histone H3 modification with >99% localization probability)

  • α-32P-ATP incorporation assays for quantitative measurement

  • Targeted mutation of substrate residues to confirm modification sites (e.g., S10A mutation in histone H3 prevents AMPylation)

• AMPylation assay:

  • Reaction buffer: 50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 150 mM NaCl

  • Substrate (e.g., recombinant BiP or histone H3 peptides): 1-5 μM

  • ATP: 100-500 μM

  • Enzyme: 0.1-1 μM

  • Incubate at 30°C for 30-60 minutes

• DeAMPylation assay:

  • First AMPylate substrates using E66G mutant

  • Purify AMPylated substrates

  • Incubate with wild-type FICD in similar buffer conditions

  • Monitor AMP release with coupled enzyme assays or loss of anti-AMP signal

• Dimerization analysis:

  • Size exclusion chromatography to determine oligomeric state

  • Crosslinking studies to stabilize transient dimers

  • Correlate oligomeric state with predominant enzymatic activity

These approaches allow for systematic analysis of both activities under controlled conditions.

How should I design control experiments when studying FICD-mediated modification of target proteins?

Robust control experiments are essential for validating FICD-mediated modifications:

• Enzyme controls:

  • Catalytically dead mutant (H205A): Should show no AMPylation activity

  • Deregulated mutant (E66G): Should show enhanced AMPylation

  • Heat-inactivated enzyme: Denatured control

  • No enzyme reaction: Control for non-enzymatic AMP addition

• Substrate controls:

  • Mutated target residues: For example, S10A mutation in histone H3 prevents modification

  • Unrelated proteins: To confirm target specificity

  • Truncated/fragmented substrates: To define minimal recognition elements

• Reaction condition controls:

  • No ATP control: Confirms ATP-dependence

  • Alternative nucleotides (GTP, CTP): Tests nucleotide specificity

  • Varying divalent cation concentrations: Determines optimal Mg²⁺ requirements

  • pH range testing: Determines pH optimum and physiological relevance

• Validation approaches:

  • Multiple detection methods: Anti-AMP antibody, mass spectrometry, and radioisotope approaches

  • Recapitulation in cellular systems with overexpression/knockdown

  • Comparison with established FICD substrates (BiP)

  • In vitro versus in vivo modification patterns

A systematic approach to controls ensures that observed modifications are specific and enzymatically driven rather than artifacts.

How can I investigate the role of dimerization in regulating FICD's enzymatic switch between AMPylation and deAMPylation?

Dimerization appears crucial for regulating FICD activity, similar to human FICD where the dimer interface links to the active site, transferring rigidity toward the inhibitory helix, preventing AMPylation and favoring deAMPylation . To investigate this regulatory mechanism:

• Structure-guided mutagenesis:

  • Identify potential dimer interface residues through homology modeling with human FICD

  • Create point mutations designed to disrupt dimerization without affecting catalytic activity

  • Generate obligate dimers through introduction of disulfide bridges or chemical crosslinkers

• Dimerization analysis:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation to determine equilibrium constants

  • FRET-based assays using fluorescently labeled FICD to monitor dimerization in real-time

  • Hydrogen-deuterium exchange mass spectrometry to map interface dynamics

• Correlation of dimerization with activity:

  • Measure AMPylation and deAMPylation activities of monomeric and dimeric forms

  • Use conditions that favor different oligomeric states (salt concentration, pH)

  • Employ rapid kinetics to capture transitional states

• Cellular studies:

  • Design mutations that affect dimerization but preserve catalytic activity

  • Assess localization and activity of dimerization mutants in cellular contexts

  • Utilize proximity ligation assays to visualize dimerization in cells

  • Correlate dimerization state with cellular stress responses

These approaches can elucidate how structural transitions regulate the functional switch between AMPylation and deAMPylation activities.

What methodologies are available to study the physiological relevance of FICD in mouse models of ER stress and disease?

To investigate FICD's physiological role in mouse models:

• Genetic approaches:

  • Conditional knockout models using Cre-lox system for tissue-specific deletion

  • Knock-in of catalytically inactive (H205A) or deregulated (E66G) mutants

  • CRISPR/Cas9-mediated generation of point mutations affecting specific activities

• ER stress models:

  • Chemical inducers: tunicamycin, thapsigargin, DTT, brefeldin A

  • Physiological stressors: high-fat diet, fasting-refeeding cycles

  • Disease models: neurodegenerative disorders, diabetes, inflammatory conditions

• Assessment parameters:

  • UPR marker analysis: PERK, IRE1α, and ATF6 pathway activation

  • Tissue damage biomarkers

  • Weight loss patterns following repeated UPR induction

  • BiP AMPylation status using anti-AMP antibodies

  • Mitochondrial function and metabolic parameters

• Experimental design considerations:

  • Acute vs. chronic stress exposure

  • Age and sex-specific effects

  • Comparison between mild physiological stress and pathological UPR induction

Based on current understanding, FICD knockout mice exhibit enhanced UPR signaling in response to short-term physiologic stress, but show more complex responses to chronic stressors like high-fat diet. Importantly, FICD appears to regulate adaptation to repeated ER stress episodes rather than initial responses, suggesting its role in maintaining tissue resiliency .

How can the crosstalk between FICD activity and other post-translational modifications be methodically investigated?

Investigating crosstalk between FICD-mediated AMPylation and other post-translational modifications (PTMs) requires a multi-faceted approach:

• PTM site mapping and competition analysis:

  • Mass spectrometry to identify and quantify different PTMs on shared substrates

  • Mutational analysis of modification sites (e.g., S10 on histone H3 can be phosphorylated or AMPylated)

  • Sequential modification assays to determine if one PTM promotes or inhibits others

  • In vitro competition assays between different modifying enzymes

• Functional consequence exploration:

  • Antibody-based detection of specific modifications in different cellular states

  • Reconstitution assays measuring functional outcomes (e.g., BiP chaperone activity)

  • Structural studies to determine how modifications affect protein conformation

  • Binding partner analysis before and after specific modifications

• Integrated proteomics approaches:

  • Enrichment strategies for AMPylated proteins combined with phosphoproteomics

  • Bioinformatic analysis to identify motifs susceptible to multiple modifications

  • Temporal dynamics studies tracking modification changes during stress responses

  • Quantitative proteomics comparing wild-type and FICD mutant systems

• Regulatory circuit analysis:

  • Inhibitor studies to block specific PTM pathways and observe effects on others

  • Genetic manipulation of PTM writers, erasers, and readers

  • Time-course experiments during cellular stress responses

  • Mathematical modeling of PTM networks

For example, investigating the relationship between AMPylation and phosphorylation at histone H3 S10 requires consideration that this site is a known target for various kinases during mitosis and gene regulation .

What are common pitfalls in detecting FICD-mediated AMPylation and how can they be addressed?

Researchers may encounter several challenges when detecting AMPylation:

• Low signal-to-noise ratio:

  • Problem: Anti-AMP antibodies may have high background or low specificity

  • Solution: Pre-clear lysates, optimize blocking conditions, use multiple antibody clones for validation, consider enrichment of AMPylated proteins before detection

• Transient modifications:

  • Problem: AMPylation may be rapidly reversed by deAMPylation activity

  • Solution: Use deAMPylation-deficient systems (E66G mutant), perform assays at lower temperatures, include phosphatase inhibitors to prevent AMP hydrolysis

• Conflicting results between in vitro and cellular systems:

  • Problem: In vitro assays may not reflect physiological conditions

  • Solution: Validate with multiple approaches, ensure proper enzyme:substrate ratios, include cellular cofactors, confirm with genetic approaches in cells

• Identification of modification sites:

  • Problem: AMP can dissociate during standard mass spectrometry procedures

  • Solution: Use electron-transfer dissociation (ETD) rather than collision-induced dissociation (CID), employ neutral loss scanning, develop targeted methods for known sites

• Distinguishing AMPylation from other adenylyl modifications:

  • Problem: AMP can be attached via different linkages (O-, N-, or S-linkage)

  • Solution: Use linkage-specific chemical approaches, enzymatic treatments with specific nucleotidases, employ high-resolution mass spectrometry

Systematic troubleshooting and method optimization can significantly improve detection reliability .

How should researchers interpret apparently contradictory data about FICD activity in different experimental systems?

When faced with contradictory FICD activity data:

• Systematic comparison of experimental conditions:

  • Enzyme concentration effects: FICD concentration can affect oligomeric state and activity ratios

  • Buffer composition differences: pH, salt, and divalent cations significantly impact activity

  • Substrate preparation variations: Different expression systems for substrates may include pre-existing modifications

• Consideration of biological context:

  • Tissue-specific effects: FICD may function differently in liver versus pancreas or other tissues

  • Stress conditions: Acute versus chronic stress exposure elicits different FICD responses

  • Environmental factors: Nutrient availability affects metabolic state and FICD function

• Technical validation approaches:

  • Cross-validate with multiple detection methods

  • Perform concentration and time-course analyses

  • Test activity under both standard and physiologically relevant conditions

  • Compare recombinant and endogenous enzyme activities

• Data integration framework:

  • Map contradictions to specific variables in experimental design

  • Consider biphasic responses and threshold effects

  • Develop testable hypotheses that could explain apparent contradictions

  • Create mathematical models incorporating regulatory switches

For example, studies have shown that FICD knockout mice exhibit enhanced UPR signaling under short-term physiologic stress but show complex responses to chronic stressors, suggesting context-dependent regulatory mechanisms .

What methodological approaches can distinguish between direct and indirect effects of FICD in complex cellular systems?

Distinguishing direct from indirect FICD effects requires:

• In vitro reconstitution:

  • Purified component analysis with recombinant proteins

  • Order-of-addition experiments to establish reaction sequence

  • Kinetic analysis to determine direct enzymatic parameters

  • Competition assays with known substrates

• Substrate trapping approaches:

  • Generate substrate-trapping mutants that form stable complexes with targets

  • Use proximity labeling methods (BioID, APEX) to identify proteins in FICD vicinity

  • Apply crosslinking strategies to capture transient interactions

  • Develop activity-based probes specific for FICD

• Genetic complementation strategies:

  • Rescue experiments with wild-type vs. catalytically inactive mutants

  • Structure-function analysis with domain-specific mutations

  • Rapid inducible systems to capture immediate versus delayed effects

  • Genetic epistasis analysis to establish pathway relationships

• Temporal dissection of responses:

  • Time-course experiments with high temporal resolution

  • Pulse-chase labeling of AMPylation events

  • Selective inhibition at different time points

  • Single-cell analysis to track response heterogeneity

For example, when studying FICD's role in the unfolded protein response, researchers should consider that FICD regulates adaptation to repeated stress rather than initial responses, indicating a complex role in maintaining tissue resilience through stress adaptation mechanisms .

What are the best approaches for quantifying the kinetics of FICD-mediated AMPylation and deAMPylation reactions?

For accurate kinetic analysis of FICD enzymatic activities:

Advanced considerations:

• Pre-steady-state kinetics:

  • Use rapid-quench flow techniques to capture transient intermediates

  • Analyze burst phase kinetics to determine rate-limiting steps

  • Employ single-turnover conditions to isolate individual reaction steps

• Regulatory factor analysis:

  • Examine effects of dimerization on kinetic parameters

  • Test allosteric modulators that might affect activity

  • Analyze substrate competition to determine preference hierarchies

• Physiological condition simulation:

  • Perform assays under crowded conditions (with PEG or Ficoll)

  • Include cellular extracts to approximate cytoplasmic environment

  • Test kinetics under various stress-mimicking conditions

• Mathematical modeling:

  • Develop ordinary differential equation models of reaction mechanisms

  • Fit experimental data to alternative mechanistic models

  • Predict system behavior under untested conditions

These approaches allow for rigorous quantitative analysis of FICD's dual enzymatic activities and their regulation.

What are promising strategies for developing specific inhibitors or modulators of FICD activity for research applications?

Developing FICD-specific modulators requires several strategic approaches:

• Structure-based drug design:

  • Crystal structure determination of mouse FICD in different functional states

  • In silico screening against the ATP binding pocket and allosteric sites

  • Fragment-based approaches to identify initial chemical scaffolds

  • Structure-activity relationship (SAR) studies of lead compounds

• High-throughput screening platforms:

  • Development of FRET-based activity assays suitable for plate format

  • Cell-based reporter systems for monitoring BiP AMPylation status

  • Phenotypic screens based on UPR readouts in FICD-dependent systems

  • Differential screening against AMPylation versus deAMPylation activities

• Allosteric modulator development:

  • Target the dimerization interface to modulate the AMPylation/deAMPylation switch

  • Identify compounds that stabilize specific conformational states

  • Screen for molecules that affect inhibitory helix positioning

  • Develop bi-specific molecules linking active site and regulatory domains

• Substrate-inspired approaches:

  • Design peptidomimetics based on BiP or histone recognition sequences

  • Develop bisubstrate analogs linking ATP and target protein recognition elements

  • Create covalent inhibitors targeting the catalytic histidine residue

  • Engineer activity-based probes for target engagement studies

The development of specific modulators would significantly advance research by allowing temporal control over FICD activity in complex biological systems.

How might researchers best explore the evolutionary conservation and divergence of FICD function across species?

To investigate evolutionary aspects of FICD function:

• Comparative genomic approaches:

  • Phylogenetic analysis of Fic domain proteins across species

  • Identification of conserved regulatory elements in FICD genes

  • Analysis of co-evolution between FICD and its target proteins

  • Examination of lineage-specific changes in FICD sequence and structure

• Functional conservation testing:

  • Cross-species complementation experiments in knockout models

  • Biochemical characterization of FICD orthologs from diverse organisms

  • Substrate specificity comparison across evolutionary distance

  • Domain swapping between orthologs to pinpoint functional divergence

• Structural biology approaches:

  • Comparative structural analysis of FICD from different species

  • Identification of conserved vs. variable surface regions

  • Analysis of dimerization interfaces across species

  • Molecular dynamics simulations to compare conformational dynamics

• Systems biology integration:

  • Network analysis of FICD-associated pathways in different organisms

  • Comparative stress response studies across model systems

  • Analysis of FICD expression patterns in tissue-specific contexts

  • Correlation of FICD function with organismal complexity

This evolutionary perspective can provide crucial insights into core conserved functions versus species-specific adaptations of FICD proteins.