Recombinant Pongo abelii GRAM domain-containing protein 1C (GRAMD1C)

What is GRAMD1C and where is it derived from?

GRAMD1C (GRAM domain-containing protein 1C) from Pongo abelii (Sumatran orangutan) is a protein involved in cellular cholesterol transport and autophagy regulation. The full-length protein contains 662 amino acids and features a characteristic GRAM domain essential for its function . The protein's amino acid sequence begins with MEGAPTVRQVMNEGDSSLATELQEDVEENPSPTVEENNVVVKKQGPNLHNWSGDWSFWIS and continues through a complex structure that enables its biological activities . This protein belongs to the GRAM family, which includes other members such as GRAMD1A, GRAMD1B, GRAMD2, and GRAMD3, all involved in various aspects of cellular lipid transport and membrane contact sites.

What is the functional significance of the GRAM domain in GRAMD1C?

The GRAM domain in GRAMD1C serves as a critical structural element that mediates its interaction with mitochondria . Research demonstrates that when the GRAM domain is deleted (ΔGRAM), GRAMD1C loses its ability to associate with mitochondrial proteins such as NDUFAF2, SHDB, and ATAD3A, as well as ER-mitochondria contact site proteins including VDAC1 and ACSL4 . This domain is therefore essential for the protein's role in facilitating cholesterol transport between cellular compartments. Experimental approaches using domain deletion mutants have confirmed that the GRAM domain determines the subcellular targeting specificity of GRAMD1C to mitochondrial membranes, similar to its yeast orthologue .

How does GRAMD1C contribute to cellular cholesterol homeostasis?

GRAMD1C plays a significant role in regulating cholesterol movement between the endoplasmic reticulum (ER) and mitochondria. When GRAMD1C is knocked out or depleted, mitochondrial cholesterol levels increase significantly, as demonstrated by both mCherry-D4 binding assays and cholesterol oxidase-based quantification . Concurrently, ER cholesterol levels decrease, evidenced by increased expression of SREBP target genes . This suggests that GRAMD1C normally facilitates cholesterol transport from mitochondria to the ER, maintaining appropriate cholesterol distribution between these organelles. The protein appears to be a negative regulator of mitochondrial cholesterol abundance, which has implications for mitochondrial membrane fluidity and function.

How does GRAMD1C regulate autophagy initiation?

GRAMD1C functions as a negative regulator of starvation-induced autophagy . Experimental evidence shows that GRAMD1C depletion significantly enhances autophagy flux during nutrient starvation. The regulatory mechanism appears to be specific to macroautophagy induced by starvation, as knockdown of GRAMD1C doesn't affect Parkin-dependent or Parkin-independent mitophagy processes . This suggests that GRAMD1C's role in autophagy regulation is pathway-specific and may be linked to its function in cholesterol transport. The modulation of autophagy by GRAMD1C likely involves altered lipid composition at autophagosome formation sites, though the precise mechanism requires further investigation through techniques such as lipidomic analysis of isolation membranes in GRAMD1C-depleted cells.

What is the relationship between GRAMD1C and mitochondrial bioenergetics?

GRAMD1C depletion leads to enhanced mitochondrial bioenergetics, specifically increasing ATP-production linked respiration and maximal respiratory capacity . Seahorse XF Analyzer measurements have confirmed this effect in both U2OS cells and ccRCC 786-O cells, indicating a conserved relationship across different cell types . Interestingly, this increased respiratory capacity occurs without significant changes to OXPHOS protein levels, mitochondrial membrane potential, or total cellular reactive oxygen species (ROS) . The enhancement of mitochondrial function appears to be linked to increased mitochondrial cholesterol levels resulting from GRAMD1C depletion. This suggests that GRAMD1C serves as a fine-tuning mechanism for mitochondrial energy production through its regulation of cholesterol transport.

How does GRAMD1C interact with other GRAM family proteins?

GRAMD1C forms heteromeric complexes with other GRAM family proteins including GRAMD1A, GRAMD1B, GRAMD2, and GRAMD3, as demonstrated by interactome studies . These interactions potentially create functional networks that collectively regulate cellular cholesterol distribution. Expression analysis in ccRCC samples reveals a weak negative correlation between GRAMD1C expression and the levels of GRAMD1A and GRAMD1B . This antagonistic relationship is further reflected in their differential influence on patient survival in ccRCC, where high GRAMD1C expression is beneficial while high GRAMD1A and GRAMD1B expression is detrimental . These findings suggest complex regulatory relationships between GRAM family members that may influence cellular metabolism and disease progression.

What methods are most effective for studying GRAMD1C localization and interactions?

To study GRAMD1C localization and interactions, researchers should employ multi-faceted approaches:

• Fluorescence microscopy: Using GFP-tagged or mCherry-tagged GRAMD1C constructs to visualize subcellular localization, particularly at ER-mitochondria contact sites.

• Domain deletion analysis: Creating GRAMD1C constructs lacking specific domains (especially ΔGRAM variants) to determine their role in localization and function .

• Co-immunoprecipitation: Identifying interaction partners through pull-down assays followed by mass spectrometry analysis, as demonstrated in the research showing GRAMD1C interactions with mitochondrial proteins .

• Proximity labeling techniques: Using BioID or APEX2 fusion proteins to identify proteins in close proximity to GRAMD1C in living cells, providing insight into its dynamic interaction network.

• Super-resolution microscopy: Employing techniques such as STORM or PALM to visualize GRAMD1C at membrane contact sites with nanometer precision.

These approaches should be used in combination to generate a comprehensive understanding of GRAMD1C's cellular interactions and functional domains.

How can researchers accurately measure mitochondrial cholesterol in GRAMD1C studies?

Researchers can employ several complementary techniques to quantify mitochondrial cholesterol levels when studying GRAMD1C:

• mCherry-D4 binding assay: Using recombinant mCherry-tagged cholesterol binding domain of Perfringolysin O (mCherry-D4) on isolated mitochondria, followed by flow cytometry or fluorescence microscopy quantification . This approach allows selective detection of accessible cholesterol on mitochondrial membranes.

• Cholesterol oxidase-based quantification: Enzymatic assays that directly measure cholesterol content in isolated mitochondrial fractions .

• Filipin staining: A fluorescent polyene antibiotic that binds to cholesterol, allowing visualization and semi-quantitative analysis of cholesterol distribution.

• Lipidomic analysis: Mass spectrometry-based identification and quantification of cholesterol and its derivatives in purified mitochondrial fractions.

• Control experiments: Including treatments with methyl-β-cyclodextrin (MBCD) to deplete cholesterol as a negative control .

Proper mitochondrial isolation and purity assessment are critical for accurate results, as contamination from other organelles can significantly skew measurements.

What approaches should be used to assess GRAMD1C's impact on autophagy?

To comprehensively evaluate GRAMD1C's influence on autophagy, researchers should implement these methodological approaches:

• LC3 flux assays: Monitoring LC3-I to LC3-II conversion with and without lysosomal inhibitors (such as Bafilomycin A1) in GRAMD1C-depleted versus control cells under various autophagy-inducing conditions .

• Fluorescence microscopy of autophagy markers: Tracking GFP-LC3 or mCherry-GFP-LC3 puncta formation and autophagic flux in living cells.

• Selective autophagy assessment: Using specific substrates to differentiate between general autophagy and selective processes like mitophagy (using mt-Keima or mito-QC reporters) .

• WIPI2 puncta quantification: Measuring early autophagosome formation sites to assess initiation events.

• Electron microscopy: Ultrastructural analysis of autophagosome and autolysosome formation.

• Biochemical analysis of autophagy signaling: Examining phosphorylation states of ULK1, AMPK, and mTOR to understand the regulatory mechanisms.

These approaches should be applied under different autophagy-inducing conditions (starvation, rapamycin treatment, etc.) to fully characterize GRAMD1C's role in various autophagy pathways.

How is GRAMD1C expression linked to cancer progression and patient outcomes?

GRAMD1C expression demonstrates significant associations with cancer progression and patient survival:

These findings indicate that GRAMD1C may serve as a potential prognostic biomarker in certain cancers, particularly ccRCC, and might represent a therapeutic target due to its role in regulating cellular metabolism and autophagy.

How do GRAMD1C and other GRAM family proteins differentially impact cancer progression?

GRAM family proteins demonstrate contrasting impacts on cancer progression and patient outcomes:

This antagonistic relationship between GRAMD1C and certain family members (GRAMD1A, GRAMD1B) suggests they may regulate opposing pathways in cancer development . The mechanisms likely involve differential effects on cholesterol transport, mitochondrial function, and autophagy regulation. The negative correlation between GRAMD1C expression and GRAMD1A/GRAMD1B levels in tumor samples further supports their potentially opposing roles in cancer biology . Understanding these differential impacts could lead to more precise prognostic markers and therapeutic strategies targeting specific GRAM family members based on tumor characteristics.

What methodological approaches should be used to study GRAMD1C in cancer models?

Researchers investigating GRAMD1C in cancer contexts should employ these methodological approaches:

• Gene expression analysis: Examining GRAMD1C expression levels across tumor stages, grades, and molecular subtypes using RNA-seq or qPCR, with correlation to clinical outcomes .

• Co-expression network analysis: Identifying genes co-expressed with GRAMD1C to elucidate functional networks, as demonstrated in ccRCC where GRAMD1C co-expression with mitochondrial genes was identified .

• Functional studies in cancer cell lines: Using CRISPR/Cas9 knockout or siRNA knockdown of GRAMD1C in relevant cancer cell lines (e.g., 786-O for ccRCC) to assess effects on proliferation, migration, invasion, and metabolic parameters .

• Metabolic profiling: Employing Seahorse XF Analyzer to measure oxygen consumption rates and extracellular acidification rates in GRAMD1C-modified cancer cells .

• Xenograft models: Evaluating the impact of GRAMD1C modulation on tumor growth, metabolism, and treatment response in vivo.

• Patient-derived organoids: Establishing 3D culture models from patient samples with various GRAMD1C expression levels to study drug responses and cellular behaviors.

• Immunohistochemistry: Analyzing GRAMD1C protein expression in tumor tissue microarrays correlated with clinicopathological parameters and immune cell infiltration.

These approaches provide complementary insights into GRAMD1C's role in cancer biology and its potential as a therapeutic target or biomarker.