Recombinant Mouse GRAM domain-containing protein 4 (Gramd4)

What is GRAM domain-containing protein 4 (Gramd4)?

GRAM domain-containing protein 4 (Gramd4) is a protein that belongs to the GRAM domain family. In humans, the orthologous protein GRAMD4 is also known as Death-Inducing Protein (DIP) and functions as a mitochondrial effector of E2F1-induced apoptosis . The protein contains a characteristic GRAM domain, which is structurally similar to pleckstrin homology (PH) domains and plays a crucial role in membrane targeting. The functional characterization of mouse Gramd4 builds upon knowledge derived from human GRAMD4 and other GRAM domain-containing proteins in the same family.

What is the functional significance of the GRAM domain in Gramd4?

The GRAM domain in Gramd4, like in other GRAM domain-containing proteins, likely serves as a critical determinant for membrane contact site (MCS) localization. Research on related GRAM domain proteins has demonstrated that this domain is required for targeting to membranes . For instance, variants of other GRAM domain proteins lacking their GRAM domain exhibit diffuse endoplasmic reticulum localization and are not observed in focal structures at membrane contact sites . In Gramd4 specifically, the GRAM domain likely contributes to its ability to interact with phospholipids and potentially detect accessible cholesterol, similar to other family members like GRAMD1b .

How does Gramd4 differ from other GRAM domain-containing proteins?

While all GRAM domain-containing proteins share structural similarities, they have specialized functions. Unlike GRAMD1a/b/c and GRAMD2/3, which primarily function at endoplasmic reticulum-plasma membrane (ER-PM) contact sites , Gramd4 appears to have distinct roles in apoptosis and tumor suppression. The human ortholog GRAMD4 has been shown to function as a p53-independent proapoptotic protein . Additionally, GRAMD4 has been implicated in inhibiting tumor metastasis through interactions with TAK1 (transforming growth factor β-activated kinase 1) and the recruitment of ITCH (itchy E3 ubiquitin protein ligase) , a function that likely extends to mouse Gramd4 but would require specific confirmation through comparative studies.

What are the recommended methods for expressing recombinant mouse Gramd4?

Based on protocols used for other GRAM domain proteins, recombinant mouse Gramd4 can be expressed using bacterial expression systems. A suggested protocol involves:

• Cloning the Gramd4 sequence (excluding the transmembrane domain for soluble protein production) into a bacterial expression vector such as pET15b with a His6-tag.

• Transforming the construct into BL21 E. coli containing the RIPL plasmid for expression of nonabundant tRNAs.

• Growing cultures at 37°C to OD600 0.7 and inducing expression with 0.5 mM IPTG for 2-3 hours.

• Harvesting cells by centrifugation and resuspending in buffer (50 mM Hepes, pH 8, 500 mM NaCl, with protease inhibitors and reducing agents).

• Lysing cells using a microfluidizer and removing insoluble material by high-speed centrifugation.

• Purifying the recombinant protein using nickel affinity chromatography with appropriate washing and elution buffers .

How can researchers assess the lipid-binding properties of recombinant Gramd4?

To characterize the lipid-binding properties of recombinant Gramd4, liposome-binding assays can be employed following these methodological steps:

• Prepare liposomes with different phospholipid compositions, including various concentrations of phosphoinositides like PI(4)P and PI(4,5)P2.

• Incubate purified recombinant Gramd4 (1-2 μM) with liposomes (approximately 1.2 mg/mL) at room temperature for 90 minutes.

• Separate bound and unbound protein by ultracentrifugation at 40,000 g for 30 minutes at 4°C.

• Analyze the supernatant and pellet fractions by SDS-PAGE and western blotting using anti-His antibodies to detect recombinant His-tagged Gramd4.

• Quantify binding using imaging systems such as LI-COR Odyssey .

This approach allows researchers to determine the lipid specificity of Gramd4's GRAM domain and compare it with other family members, which have shown preference for phosphoinositides and potentially accessible cholesterol.

What techniques are available for studying Gramd4 localization in cells?

Several microscopy techniques can be employed to study Gramd4 localization:

• Spinning disk microscopy: Using fluorescently labeled Gramd4 (e.g., Gramd4-eGFP) expressed in mammalian cells alongside markers for different cellular compartments.

• Z-stack imaging and reconstruction: To analyze the three-dimensional localization pattern of Gramd4 in relation to other cellular structures.

• Total Internal Reflection Fluorescence (TIRF) microscopy: Particularly useful if Gramd4 localizes near the plasma membrane, allowing selective illumination within ~100 nm of the plasma membrane.

• Co-localization analysis: Quantifying the extent of overlap between Gramd4 and other cellular markers using pixel-based co-localization measurements .

For optimal results, these studies should include appropriate controls such as Gramd4 variants lacking the GRAM domain to confirm domain-specific localization patterns.

How is Gramd4 implicated in cancer biology?

Research on human GRAMD4 suggests that mouse Gramd4 may have important tumor-suppressive functions. GRAMD4 expression has been found to be lower in hepatocellular carcinoma (HCC) samples compared to normal tissues, with reduced expression correlating with worse prognosis for patients after surgical resection . Functionally, GRAMD4 has been demonstrated to inhibit cancer cell migration, invasion, and metastasis. The mechanisms involve GRAMD4 interaction with TAK1, promoting its protein degradation and resulting in the inactivation of MAPK and NF-κB signaling pathways . Given the high conservation of these pathways between humans and mice, similar functions would be expected for mouse Gramd4, though specific studies would be needed to confirm this extrapolation.

What is the relationship between Gramd4 and apoptosis pathways?

Gramd4, like its human ortholog, is likely involved in apoptotic pathways. Human GRAMD4 has been characterized as a p53-independent proapoptotic protein , functioning as a mitochondrial effector of E2F1-induced apoptosis . This suggests that mouse Gramd4 may play important roles in programmed cell death that do not require p53 activation. Researchers investigating apoptotic mechanisms should consider examining Gramd4's interaction with mitochondrial proteins and its effects on mitochondrial membrane permeability. Additionally, its relationship with E2F1-regulated pathways would be important to characterize in mouse models to establish cross-species conservation of these functions.

How can Gramd4 be utilized to study membrane contact sites and lipid transport?

Building on knowledge from other GRAM domain-containing proteins, Gramd4 could serve as a valuable tool for studying membrane contact sites and lipid transport:

• Generate fluorescently tagged Gramd4 constructs to visualize potential membrane contact sites in live cells

• Create domain-deletion variants to assess the contribution of different protein regions to membrane targeting

• Combine with lipid biosensors to study co-localization with specific lipid pools

• Perform proximity labeling experiments (BioID or APEX) with Gramd4 to identify protein interaction partners at membrane contact sites

• Develop Gramd4 GRAM domain-based biosensors, similar to the GRAM-W sensor derived from GRAMD1b , to potentially detect specific lipid compositions

The GRAM domains of related proteins function as coincidence detectors for specific lipids; determining whether Gramd4's GRAM domain has similar properties could yield valuable insights into specialized membrane domains.

What are the considerations for generating Gramd4 knockout mouse models?

When designing Gramd4 knockout mouse models, researchers should consider:

• Strategy selection: Decide between conventional knockout, conditional knockout, or knockin approaches based on research questions

• Targeting approach: Design CRISPR-Cas9 or homologous recombination strategies that avoid affecting neighboring genes

• Validation methods: Plan comprehensive validation including genomic PCR, RT-PCR, western blotting, and immunohistochemistry

• Phenotypic analysis: Based on known functions, focus on:

  • Apoptotic responses in various tissues

  • Tumor susceptibility and metastasis models

  • Lipid metabolism and distribution

  • Interaction with E2F1-dependent pathways

  • Potential developmental defects if Gramd4 has essential functions

• Compensatory mechanisms: Assess potential upregulation of other GRAM domain-containing proteins that might compensate for Gramd4 loss

How does post-translational modification affect Gramd4 function?

Although specific information about post-translational modifications (PTMs) of mouse Gramd4 is limited in the provided search results, researchers should investigate potential PTMs as they likely regulate Gramd4's function:

• Phosphorylation: Examine how kinase pathways might regulate Gramd4's activity or localization

• Ubiquitination: Given GRAMD4's reported interaction with the E3 ubiquitin ligase ITCH , investigate both how Gramd4 might be regulated by ubiquitination and how it might influence the ubiquitination of other proteins

• Other modifications: Consider glycosylation, SUMOylation, or acetylation as potential regulatory mechanisms

• Methodology: Implement mass spectrometry-based proteomics approaches to identify PTMs under different cellular conditions

• Functional impact: Generate point mutations at identified modification sites to assess their impact on Gramd4's localization, interaction partners, and cellular functions

How conserved is Gramd4 across species?

Phylogenetic analysis indicates that GRAM domain-containing proteins form a conserved family anchored by the presence of the GRAM domain . Researchers should consider:

• Performing comparative sequence analysis to determine conservation levels of different Gramd4 domains

• Examining whether functional properties are conserved between mouse Gramd4 and human GRAMD4

• Conducting complementation experiments to test whether mouse Gramd4 can rescue phenotypes in human cell lines with GRAMD4 knockdown

• Investigating potential species-specific interaction partners that might influence Gramd4 function in different organisms

What is the relationship between Gramd4 and other GRAM domain-containing proteins in experimental design?

When designing experiments involving Gramd4, researchers should consider its relationship with other GRAM domain-containing proteins:

• Include appropriate controls with other GRAM domain proteins (GRAMD1a/b/c, GRAMD2, GRAMD3) to distinguish specific vs. general GRAM domain functions

• Consider potential functional redundancy - knockdown/knockout of multiple family members might be necessary to observe certain phenotypes

• Examine tissue-specific expression patterns to identify contexts where Gramd4 might have unique or predominant functions

• Design domain-swapping experiments to determine which protein regions confer specific functional properties

The fact that GRAMD1a and GRAMD2a mark distinct ER-PM contact sites with different lipid dependencies suggests specialized functions among family members that should be considered when studying Gramd4.