Recombinant Human GTPase IMAP family member 2 (GIMAP2)

What is the structural organization of the GIMAP2 protein?

GIMAP2 belongs to the GTPase of immunity-associated protein (GIMAP) family and features a distinctive structure among GIMAPs. The protein consists of:

• A core G domain of the TRAFAC (translation factor associated) class

• An additional helix α3* between strand 5 and helix α4

• A C-terminal extension of two helices (α6 and α7)

• Two C-terminal hydrophobic segments that distinguish it from other human GIMAPs

The G domain contains guanine nucleotide-binding motifs G1-G5 with sequence differences compared to canonical TRAFAC GTPases. Notably, GIMAP2 features an amphipathic helix α7 connected to the G domain by a 16-residue disordered linker, which directly contacts the switch II region .

How does GIMAP2 interact with nucleotides?

GIMAP2 binds GTP with high affinity (Kd of 40 nM) and GDP with lower affinity (Kd of 630 nM). The nucleotide-binding mechanism involves:

• The G1-G5 motifs function similarly to other GTPases despite sequence differences

• A histidine in the second position of the G4 motif (replacing the usual lysine) recognizes guanine via π-π stacking interactions

• Asp80 in the G3 motif points toward the β-phosphate, whereas in most other TRAFAC GTPases, a glycine acts as γ-phosphate sensor

• The catalytic site lacks the glutamine/histidine residue common in Ras-like GTPases, replaced by a hydrophobic residue (Met81)

Notably, GIMAP2 shows extremely low GTPase activity, with no detectable GTP hydrolysis even during long incubations (>24 h) and at high protein concentrations (50 μM) .

What expression patterns does GIMAP2 show in human tissues?

GIMAP2 expression is primarily restricted to:

• T cells and other lymphocytes

• Blood cells, including platelets

• Spleen and other lymphoid tissues

According to BioGPS data, GIMAP2 shows limited expression in non-immune tissues. In pathological conditions, GIMAP2 expression patterns differ:

• Maintained in human lymphoma T cell lines when other GIMAP family members are downregulated

• Significantly upregulated in oral squamous cell carcinoma (OSCC) compared to normal tissues

What are recommended approaches for expressing and purifying recombinant GIMAP2?

For successful GIMAP2 recombinant protein production:

• Expression system selection:

  • Mammalian expression systems (e.g., HEK293) for full-length protein with post-translational modifications

  • E. coli expression for truncated constructs (e.g., GIMAP2 1-234, lacking C-terminal hydrophobic regions)

• Purification strategy:

  • Affinity chromatography using His-tag or GST-tag fusion proteins

  • Size exclusion chromatography to separate oligomeric states

  • Ion-exchange chromatography for final polishing

• Stability considerations:

  • Include GTP or non-hydrolyzable GTP analogs to stabilize protein conformation

  • Avoid detergents that may disrupt the amphipathic helix α7

For example, Schwefel et al. successfully expressed and purified various GIMAP2 constructs including GIMAP2 1-260 and GIMAP2 1-234 for crystallographic studies .

How can I assess GIMAP2 expression levels in patient samples or cell lines?

Several validated approaches for GIMAP2 detection include:

• RT-qPCR analysis:

  • Design primers specific to GIMAP2 (avoid cross-reactivity with other GIMAP family members)

  • Normalize expression to established housekeeping genes

  • Example from HCC research: GIMAP mRNA levels measured in tumor vs. non-tumor tissues

• Immunodetection methods:

  • Western blotting: Use specific antibodies against GIMAP2

  • Immunohistochemistry (IHC): For tissue localization and expression pattern analysis

  • ELISA: For quantification in serum or blood samples

• Comparison approach:

  • Always include matched non-tumor/normal tissue controls

  • Include positive controls (lymphoid tissues) where GIMAP2 is highly expressed

  • Consider using blood samples as an accessible source for GIMAP2 expression analysis

How can I visualize GIMAP2 cellular localization?

For accurate subcellular localization:

• Fluorescent protein tagging:

  • Express GIMAP2 with mCherry/GFP tag (N-terminal tagging recommended)

  • Transfect into relevant cell lines (e.g., Jurkat T cells as used by Schwefel et al.)

  • GIMAP2 localizes to spherical structures (~0.8 μm diameter) identified as lipid droplets

• Co-localization analysis:

  • Use BODIPY 493/503 to stain lipid droplets

  • Test against other organelle markers (mitochondria, ER, lysosomes) to exclude alternative localizations

  • GIMAP2 signal enriches at the phospholipid monolayer surrounding lipid droplets

• Deletion construct analysis:

  • Create constructs lacking C-terminal hydrophobic segments to confirm targeting mechanism

  • Express hydrophobic segments alone fused to fluorescent proteins to verify sufficiency for localization

What methods are effective for studying GIMAP2 oligomerization?

Based on published research, effective approaches include:

• Analytical ultracentrifugation (AUC):

  • Analyze assembly status at different protein concentrations

  • Compare GDP-bound vs. GTP-bound states (GIMAP2 dimerizes with low affinity only with GTP)

  • Test mutants affecting specific interfaces (e.g., S54A, R117D in G interface)

• Analytical gel filtration:

  • Monitor oligomeric state changes in solution

  • Compare different truncation constructs (e.g., GIMAP2 1-234 vs. GIMAP2 1-223)

  • Assess effects of nucleotide binding on oligomerization

• Crystallographic analysis:

  • Crystallize GIMAP2 in different nucleotide-loading states

  • Identify interfaces mediating oligomerization (G interface and C interface)

  • Validate through structure-guided mutagenesis

• Mutagenesis studies:

  • Target key residues in oligomerization interfaces (R117D in G interface, R224D in C interface)

  • Test functional consequences in cellular models (e.g., effects on lipid droplet formation)

How does GIMAP2 expression correlate with cancer progression?

Research indicates contrasting GIMAP2 expression patterns in different cancers:

• Oral Squamous Cell Carcinoma (OSCC):

  • Significantly upregulated in OSCC-derived cell lines and primary specimens

  • Higher expression correlates with tumor progression

  • Knockdown decreases cell growth by affecting cell cycle regulators (CDK4, CDK6, phosphorylated Rb)

  • GIMAP2 appears to inhibit apoptosis by upregulating Bcl-2 and downregulating Bax and Bak

• Lymphoma:

  • GIMAP2 expression maintained in human lymphoma T cell lines when other GIMAP family members are downregulated

  • Suggests a role in cancer cell survival

• Hepatocellular Carcinoma (HCC):

  • Other GIMAP family members (GIMAP5, GIMAP6) are downregulated

  • GIMAP2-specific expression in HCC is not well characterized

• Lung Adenocarcinoma (LUAD):

  • GIMAP family members show altered expression patterns

  • Systematic analysis needed to fully characterize GIMAP2's role

How can I design experiments to assess GIMAP2's role in apoptosis and cell survival?

Based on current research, recommended approaches include:

• Gene knockdown/knockout studies:

  • Use siRNA or CRISPR-Cas9 to target GIMAP2

  • Measure effects on:

    • Cell proliferation (e.g., MTT/XTT assays, BrdU incorporation)

    • Cell cycle progression (flow cytometry)

    • Apoptosis markers (Annexin V/PI staining, caspase activation assays)

• Protein interaction studies:

  • Investigate interactions with Bcl-2 family members

  • Co-immunoprecipitation with anti-apoptotic (Bcl-2) and pro-apoptotic (Bax, Bak) proteins

  • Test if interactions are enhanced in presence of membranes/lipids

• Pathway analysis:

  • Monitor effects of GIMAP2 manipulation on:

    • Cell cycle regulators (CDK4, CDK6, p53, p21)

    • Phosphorylation status of Rb protein

    • Expression of pro- and anti-apoptotic factors

• Rescue experiments:

  • Re-express wild-type or mutant GIMAP2 in knockdown cells

  • Test specific domain functions using truncation mutants

  • Assess nucleotide-binding mutants to determine GTP-dependency

How do GIMAP2's two distinct interfaces mediate oligomerization and what are their functional implications?

GIMAP2 oligomerization involves two separate interfaces with distinct properties:

• G interface (GTP-dependent):

  • Forms across the guanine nucleotide-binding site

  • Only observed in GTP-bound form, not GDP-bound state

  • Approximately 600 Ų surface area

  • Involves conserved box, switch I, G4 motif, and helix α3*

  • Key interactions:

    • Arg117 hydrogen bonds to Gln114 of opposing monomer

    • Guanine base directly participates via hydrogen bond to Asp150

    • GTP sensing mediated by switch I stabilization

  • Mutation of S54A or R117D prevents GTP-dependent dimerization

  • Low-affinity interaction (Kd = 250 μM) typical for membrane-associated proteins

• C interface (nucleotide-independent):

  • Formed at C-terminus when helix α7 is absent

  • Present in both GDP- and GTP-bound states

  • Larger interface (~1100 Ų), mostly hydrophobic

  • Involves helices α2 of switch II, α3, and α6

  • Removal of α7 enables stable dimerization via this interface

  • Further shortening of helix α6 (GIMAP2 1-223) disrupts this interface

  • Similar behavior observed in GIMAP5, suggesting conserved mechanism

Functional implications:

• GTP binding triggers conformational changes in switch II

• These changes disrupt contacts between G domain and helix α7

• On membrane surfaces, concentration effects enhance low-affinity interactions

• GIMAP2 oligomers at lipid droplet surfaces promote lipid droplet formation

• Oligomerization mutants (R117D, R224D) localize to lipid droplets but don't increase lipid droplet numbers

What experimental approaches can assess the functional connection between GIMAP2 and lipid metabolism?

To investigate GIMAP2's role in lipid metabolism:

• Lipid droplet quantification:

  • Express wild-type or mutant GIMAP2 in relevant cell lines

  • Stain lipid droplets with BODIPY 493/503 or other neutral lipid dyes

  • Quantify number and size of lipid droplets per cell

  • Compare effects of oligomerization-deficient mutants

• Lipid composition analysis:

  • Extract lipids from cells with/without GIMAP2 overexpression

  • Perform lipidomics using mass spectrometry

  • Analyze changes in neutral lipid content and phospholipid composition

  • Test if GIMAP2 affects specific lipid species

• Protein-membrane interaction assays:

  • Assess binding of purified GIMAP2 to artificial membranes

  • Use liposomes with different lipid compositions

  • Determine if interaction is enhanced by GTP binding

  • Test role of amphipathic helix α7 in membrane association

• Co-localization with lipid metabolism machinery:

  • Assess co-localization with proteins involved in lipid droplet biogenesis

  • Investigate potential interactions with:

    • DGAT1/2 (diacylglycerol acyltransferases)

    • Perilipins and other lipid droplet-associated proteins

    • ER-lipid droplet contact site proteins

• Functional assays:

  • Measure fatty acid uptake and triglyceride synthesis rates

  • Assess lipid droplet turnover through pulse-chase experiments

  • Investigate effects under lipid-loading conditions or cellular stress

What are common challenges in working with recombinant GIMAP2 and how can they be addressed?

Researchers frequently encounter these challenges:

• Protein solubility issues:

  • Challenge: Full-length GIMAP2 contains hydrophobic regions that reduce solubility

  • Solution: Express truncated constructs lacking C-terminal hydrophobic segments

  • Alternative: Use mild detergents or lipid nanodiscs for full-length protein

• Oligomerization heterogeneity:

  • Challenge: Multiple oligomeric species complicate structural and functional studies

  • Solution: Lock protein in specific states using mutations (e.g., R117D to prevent G interface dimerization)

  • Alternative: Separate oligomeric species by size exclusion chromatography before experiments

• Low GTPase activity:

  • Challenge: GIMAP2 shows negligible GTP hydrolysis in vitro

  • Solution: Use non-hydrolyzable GTP analogs (GTPγS, GMPPNP) to maintain GTP-bound state

  • Alternative: Investigate if cellular factors enhance GTPase activity

• Expression level variability:

  • Challenge: Inconsistent expression in different cell types

  • Solution: Optimize codon usage for expression system

  • Alternative: Use inducible expression systems with careful titration

How can I reconcile contradictory findings about GIMAP family proteins in different cancer types?

To address contradictory findings regarding GIMAP expression patterns:

• Consider tissue-specific functions:

  • Different GIMAP members may have tissue-specific roles

  • GIMAP2 is upregulated in OSCC but other GIMAPs are downregulated in HCC

  • Design studies comparing multiple GIMAP members in the same samples

• Account for methodological differences:

  • Variation in detection methods (antibody specificity, primer design)

  • Differences in sample processing and normalization

  • Standardize protocols when comparing across studies

• Analyze larger datasets:

  • Utilize public databases (TCGA, GEO) to validate findings

  • Perform meta-analyses of multiple studies

  • Use integrated approaches combining transcriptomics, proteomics, and functional data

• Consider disease stage and heterogeneity:

  • Document disease progression stage in all samples

  • Account for tumor heterogeneity through single-cell approaches

  • Correlate GIMAP expression with clinical parameters and outcomes

What are promising new approaches to investigate GIMAP2's role in immune function?

Emerging technologies and approaches include:

• Single-cell analysis:

  • Apply scRNA-seq to identify cell-specific expression patterns

  • Use CyTOF or spectral flow cytometry to correlate GIMAP2 with immune cell states

  • Combine with functional readouts to link expression to cellular activities

• Interactome mapping:

  • Employ BioID or APEX proximity labeling to identify interaction partners

  • Focus on membrane-proximal interactions at lipid droplet surfaces

  • Investigate potential interactions with immune signaling complexes

• Conditional knockout models:

  • Generate tissue-specific or inducible GIMAP2 knockout systems

  • Focus on lymphocyte development and function

  • Assess effects on immune responses to various challenges

• Structure-based drug design:

  • Utilize the solved crystal structure for in silico screening

  • Develop small molecules targeting the G interface or nucleotide binding pocket

  • Create tools to modulate GIMAP2 function in research and therapeutic contexts

How might the evolutionary relationships between GIMAPs and related GTPases inform functional studies?

Evolutionary insights suggest several research directions:

• Comparative structural analysis:

  • GIMAP2 shares structural features with septins and dynamin-like GTPases

  • The G interface of GIMAP2 shows striking similarity to the dynamin G domain dimer

  • Investigate if mechanisms of membrane association are conserved

• Functional conservation testing:

  • Examine if GIMAP2's role in lipid droplet biology is related to membrane remodeling activities of dynamins

  • Test if GIMAP2 affects membrane curvature or dynamics

  • Investigate potential roles in membrane contact sites or organelle interactions

• Cross-family functional analysis:

  • Compare GIMAP2 with related proteins lacking orthologues in mice (human-specific functions)

  • Contrast with GIMAP5 (has rodent orthologues) to separate conserved vs. species-specific functions

  • Utilize insights from plant GIMAP proteins to identify core functional mechanisms

• Evolutionary adaptation analysis:

  • Investigate selective pressures on different GIMAP family members

  • Identify rapidly evolving regions that might mediate pathogen interactions

  • Explore potential roles in host-pathogen evolutionary arms races