Recombinant Drosophila erecta Adenosine monophosphate-protein transferase FICD homolog (GG10411)

Basic Research Questions

• What is Drosophila erecta Adenosine monophosphate-protein transferase FICD homolog (GG10411)?

  Drosophila erecta Adenosine monophosphate-protein transferase FICD homolog (GG10411) is a full-length protein found in D. erecta fruit flies that functions as an adenosine monophosphate-protein transferase. It belongs to the FICD (FIC domain-containing) protein family, which is involved in post-translational modification of target proteins through AMPylation (the addition of AMP to proteins). The protein has 495 amino acids and shares significant sequence homology with FICD proteins in other Drosophila species such as D. willistoni and D. yakuba . Functionally, it may play roles in cellular stress responses, protein homeostasis, and potentially lifespan regulation through connections to AMP-related metabolic pathways.

• How does GG10411 compare to FICD homologs in other Drosophila species?

  GG10411 in D. erecta shares significant sequence conservation with FICD homologs in other Drosophila species, though with species-specific variations. The protein has 495 amino acids, compared to 498 in D. willistoni (GK14760) and 495 in D. yakuba (GE13868) . Sequence analysis shows conserved functional domains across species, particularly in the catalytic core regions responsible for AMPylation activity. The conservation pattern suggests evolutionary pressure to maintain FICD functionality while allowing some adaptation to species-specific requirements. Cross-species comparative analysis provides insights into which protein regions are most critical for function versus those that may be involved in species-specific adaptations of the protein.

• What expression systems are typically used for producing recombinant GG10411?

  Recombinant GG10411 is typically produced in prokaryotic expression systems, predominantly E. coli, similar to the approach used for other Drosophila FICD homologs . The standard methodology involves:

  • Cloning the full-length gene (coding for amino acids 1-495) into a suitable expression vector

  • Adding an N-terminal His-tag for purification purposes

  • Expression in E. coli under optimized conditions

  • Purification using affinity chromatography

  • Final preparation as a lyophilized powder for storage stability

  The purified protein typically achieves >90% purity as confirmed by SDS-PAGE analysis. Alternative expression systems such as yeast or insect cells may be considered for specific research applications requiring eukaryotic post-translational modifications, though these are less commonly used for basic functional studies.

Experimental Design and Methodology

• What are the optimal storage and handling conditions for recombinant GG10411?

  For optimal stability and functionality of recombinant GG10411, follow these evidence-based storage and handling protocols:

  Condition	Recommendation	Rationale
  Storage form	Lyophilized powder	Maximizes stability
  Storage temperature	-20°C to -80°C	Prevents degradation
  Buffer composition	Tris/PBS-based buffer with 6% Trehalose, pH 8.0	Maintains protein integrity
  Reconstitution	Deionized sterile water to 0.1-1.0 mg/mL	Optimal concentration range
  Long-term storage	Add glycerol to 5-50% final concentration	Prevents freeze damage
  Working aliquots	Store at 4°C for up to one week	Minimizes freeze-thaw cycles

  Avoid repeated freeze-thaw cycles as they significantly reduce protein activity. After reconstitution, aliquot the protein and store unused portions at -80°C. When working with the protein, maintain cold chain integrity and use fresh aliquots for critical experiments .

• What assays are recommended for confirming the enzymatic activity of GG10411?

  To confirm the enzymatic activity of GG10411 as an adenosine monophosphate-protein transferase, researchers should employ a multi-faceted approach:

  • Primary activity assay: Monitor AMP transfer to target proteins using radiolabeled ATP (α-32P-ATP) followed by SDS-PAGE separation and autoradiography

  • Mass spectrometry validation: Identify AMPylated substrates and specific modification sites using high-resolution MS/MS analysis

  • Phosphorimaging quantification: Measure modification stoichiometry and enzyme kinetics

  • In vitro substrate screening: Test candidate proteins for modification, focusing on endoplasmic reticulum chaperones like BiP/GRP78 as these are common FICD targets

  • AMPylation-specific antibodies: Use antibodies recognizing AMP modifications for western blotting

  • Site-directed mutagenesis controls: Include catalytically inactive mutants (typically histidine to alanine mutations in the FIC domain) as negative controls

  These complementary approaches provide comprehensive validation of enzymatic activity and substrate specificity .

• How can I design experiments to investigate GG10411's role in lifespan regulation?

  When investigating GG10411's potential role in lifespan regulation, design your experiments based on established connections between AMP biosynthesis, AMPK signaling, and Drosophila longevity:

  1. Genetic manipulation approaches:

     • Generate D. erecta transgenic lines with GG10411 overexpression or knockdown

     • Create heterozygous mutations in GG10411 to examine partial loss-of-function effects

     • Use tissue-specific drivers (especially fat body and muscle) for targeted expression

  2. Lifespan analysis protocols:

     • Measure adult lifespan curves under standard conditions

     • Test survival under various stressors (oxidative, thermal, starvation)

     • Compare effects in different genetic backgrounds

  3. Metabolic measurements:

     • Quantify AMP:ATP and ADP:ATP ratios using HPLC

     • Measure AMPK activation (phosphorylation status)

     • Assess energy metabolism markers

  4. Dietary manipulations:

     • Test effects of adenine supplementation

     • Combine with dietary restriction protocols

     • Examine interactions with known lifespan-extending interventions

  This experimental framework leverages the established connection between adenosine nucleotide metabolism and Drosophila lifespan regulation . Focus measurements on metabolic tissues (fat body and muscle) where AMPK expression has demonstrated lifespan effects.

Advanced Research Applications

• How do research approaches differ when investigating post-translational modifications by GG10411 versus its role in reproductive biology?

  Research investigating GG10411 demonstrates distinct methodological approaches depending on whether the focus is post-translational modifications or reproductive biology:

  Research Focus	Post-translational Modifications	Reproductive Biology
  Primary techniques	Biochemical assays, mass spectrometry, protein interaction studies	Genetic crosses, morphological analyses, behavioral assays
  Model systems	Cell-free systems, cell culture, whole organism	Whole organism, population studies
  Key measurements	Enzyme kinetics, substrate specificity, modification sites	Fertility rates, reproductive success, genital morphology
  Genetic approaches	Site-directed mutagenesis of catalytic residues	Analysis of natural variation, genetic crosses
  Time scale	Hours to days (enzymatic reactions)	Generations (evolutionary studies)
  Relevant contexts	Cellular stress, protein homeostasis	Sexual selection, mating behavior, species isolation

  While studying enzymatic functions requires biochemical and proteomic approaches, reproductive biology investigations necessitate evolutionary and ecological perspectives, particularly considering D. erecta's unique genital morphology that influences reproductive success . Researchers should adjust experimental design accordingly, potentially examining how GG10411-mediated protein modifications might influence reproductive tissues or processes.

• What are the methodological challenges in studying the evolutionary conservation of GG10411 across different Drosophila species?

  Studying the evolutionary conservation of GG10411 across Drosophila species presents several methodological challenges that require specific technical approaches:

  1. Sequence divergence handling:

     • Use progressive multiple sequence alignment algorithms with sensitivity for divergent sequences

     • Employ structure-guided alignments when sequence identity falls below 40%

     • Implement phylogeny-aware gap penalties in alignments

  2. Functional conservation assessment:

     • Develop cross-species complementation assays

     • Use domain-swapping experiments to identify functionally conserved regions

     • Test substrate specificity across orthologs

  3. Evolutionary rate analysis:

     • Calculate dN/dS ratios to identify selection pressures

     • Use branch-site models to detect lineage-specific selection

     • Analyze coevolution between FICD and its substrates

  4. Tissue-specific expression profiling:

     • Compare expression patterns across species using RNA-seq

     • Develop cross-reactive antibodies targeting conserved epitopes

     • Use reporter constructs with orthologous promoters

  5. Structural biology challenges:

     • Express and purify orthologs using standardized protocols for comparison

     • Perform comparative structural analysis (X-ray crystallography or cryo-EM)

     • Model species-specific protein-substrate interactions

  These approaches help overcome the challenges posed by sequence divergence, structural variations, and different evolutionary pressures across Drosophila species .

• What techniques can effectively assess the interaction between GG10411 and transposable element activity in D. erecta?

  To effectively assess potential interactions between GG10411 and transposable element activity in D. erecta, researchers should implement a multi-layered experimental approach:

  1. Genomic analysis:

     • Map P-element insertions relative to GG10411 loci using ONT long-read sequencing

     • Track population frequencies of insertions across generations

     • Analyze chromatin accessibility around GG10411 in response to P-element invasion

  2. Small RNA profiling:

     • Sequence and analyze piRNA and siRNA populations targeting P-elements

     • Compare small RNA profiles between wild-type and GG10411 mutant backgrounds

     • Assess maternal deposition of regulatory RNAs

  3. Transcriptome analysis:

     • Monitor expression changes of GG10411 during P-element invasion

     • Analyze splicing patterns of P-element transcripts

     • Perform stranded RNA-Seq at regular generational intervals

  4. Functional genetics:

     • Create GG10411 knockdown/overexpression lines to test effects on transposon silencing

     • Assess transgenerational effects through carefully controlled genetic crosses

     • Identify genetic interactions with known piRNA pathway components

  This approach combines genomic, transcriptomic, and genetic techniques to comprehensively assess the relationship between GG10411 activity and transposable element regulation in experimental populations .

Specialized Research Considerations

• How can I develop an experimental system to test whether GG10411 influences the morphology of male genitalia in D. erecta?

  To investigate GG10411's potential role in D. erecta male genital morphology, develop an experimental system that integrates developmental genetics with morphological analysis:

  1. Genetic manipulation strategies:

     • Generate CRISPR/Cas9 knockouts or hypomorphic alleles of GG10411

     • Create tissue-specific knockdown using RNAi driven by genital disc-specific GAL4 drivers

     • Develop temperature-sensitive conditional alleles for temporal control

  2. Developmental analysis:

     • Perform immunohistochemistry on pupal genitalia using anti-E-cadherin to visualize apical cell junctions

     • Compare developmental trajectories between wild-type and GG10411-modified backgrounds

     • Track the development of specific structures like the postgonal process and aedeagus

  3. Morphometric analysis:

     • Use confocal microscopy to image adult male genitalia

     • Implement geometric morphometrics for quantitative shape analysis

     • Compare three-dimensional structure of processes using micro-CT scanning

  4. Functional assessment:

     • Conduct mating assays to assess reproductive success

     • Examine physical interaction with female genitalia

     • Analyze potential relationships to copulatory wounding

  This experimental system enables comprehensive analysis of how GG10411-mediated processes influence the development and final morphology of male genital structures, with particular attention to the complex three-dimensional nature of phallic processes that are critical for reproductive success in D. erecta .

• What are the best approaches for investigating potential links between GG10411, AMP biosynthesis, and stress response in D. erecta?

  To investigate the relationship between GG10411, AMP biosynthesis, and stress response in D. erecta, implement these evidence-based approaches:

  1. Metabolomic profiling:

     • Quantify adenine nucleotide pools (AMP, ADP, ATP) using targeted LC-MS/MS

     • Measure AMP:ATP ratios under normal and stress conditions

     • Track metabolic changes during stress recovery in wild-type vs. GG10411 mutant backgrounds

  2. AMPK signaling analysis:

     • Assess AMPK phosphorylation status using phospho-specific antibodies

     • Monitor downstream targets of AMPK activation

     • Compare signaling dynamics between control and GG10411-modified flies

  3. Stress resistance assays:

     • Subject flies to oxidative stress (paraquat, H₂O₂)

     • Apply thermal stress protocols (heat shock)

     • Conduct starvation resistance tests

     • Compare survival curves between experimental groups

  4. Gene expression analysis:

     • Perform RNA-seq under normal and stress conditions

     • Analyze expression of stress response genes

     • Compare transcriptional profiles between tissue types (fat body vs. muscle)

  5. Dietary manipulation:

     • Supplement diet with adenine to modify AMP biosynthesis

     • Combine with GG10411 genetic manipulation

     • Test interactions with dietary restriction protocols

  These approaches provide a comprehensive assessment of GG10411's potential role in connecting AMP metabolism to stress response mechanisms, building on established connections between adenosine nucleotide ratios and stress resistance in Drosophila .

• What controls and validation methods should be employed when studying the AMPylation activity of GG10411 in Drosophila tissues?

  When studying the AMPylation activity of GG10411 in Drosophila tissues, implement these critical controls and validation methods:

  1. Enzymatic controls:

     • Catalytic-dead mutant: Generate a histidine-to-alanine mutation in the FIC domain catalytic site as a negative control

     • Positive control: Include a well-characterized FICD protein from another species with established activity

     • Substrate controls: Test known FICD substrates (e.g., BiP/Hsp70 family proteins) alongside experimental targets

  2. Tissue-specific validation:

     • Tissue preparation controls: Compare fresh vs. fixed tissues to account for post-mortem modification changes

     • Subcellular fractionation: Validate activity in relevant compartments (typically ER-enriched fractions)

     • Cell type-specific analysis: Use FACS-sorted cells or single-cell approaches for heterogeneous tissues

  3. Analytical validation:

     • Multiple detection methods: Combine radioactive labeling, anti-AMP antibodies, and mass spectrometry

     • Site-mapping confirmation: Verify modification sites by MS/MS and site-directed mutagenesis of substrate residues

     • Quantitative standards: Include known quantities of synthetically AMPylated peptides as standards

  4. Physiological relevance:

     • Stress induction: Compare AMPylation profiles under normal vs. stress conditions

     • Developmental timing: Assess activity across developmental stages

     • In vivo vs. in vitro correlation: Validate that in vitro findings reflect in vivo modification patterns

  These rigorous controls and validation methods ensure specific attribution of AMPylation activity to GG10411 rather than other enzymes or artifacts, establishing confidence in the biological significance of findings.