Recombinant Human GRAM domain-containing protein 1C (GRAMD1C)

What is the domain organization of GRAMD1C and how does it compare to other GRAM family proteins?

GRAMD1C belongs to the GRAM domain-containing family proteins that includes GRAMD1a, GRAMD1b, GRAMD2a, and others. The typical domain structure includes:

• An N-terminal GRAM domain crucial for membrane targeting and lipid sensing

• A cholesterol-harboring StART-like domain for sterol transport

• A C-terminal transmembrane domain that anchors the protein to the endoplasmic reticulum (ER)

The GRAM domain functions as a coincidence detector, capable of sensing both accessible cholesterol and anionic lipids such as phosphatidylserine (PS) . Unlike some other family members that primarily localize to ER-PM contact sites, GRAMD1C shows significant interaction with mitochondria, mediated through its GRAM domain .

What is the primary cellular function of GRAMD1C?

GRAMD1C primarily functions as an ER-resident protein involved in non-vesicular cholesterol transport. Specifically, it:

• Facilitates cholesterol transport from the plasma membrane to the ER

• Regulates mitochondrial cholesterol levels through ER-mitochondria cholesterol transport

• Acts as a negative regulator of starvation-induced autophagy

• Contributes to cellular cholesterol homeostasis by sensing and moving accessible cholesterol pools

Research indicates that GRAMD1C interacts with mitochondria through its GRAM domain and influences mitochondrial bioenergetics by regulating cholesterol movement between the ER and mitochondria .

What methods are effective for studying GRAMD1C localization and dynamics?

Several complementary approaches can be employed to study GRAMD1C localization and dynamics:

• Fluorescence microscopy with tagged GRAMD1C variants:

  • Using GRAMD1C-eGFP to visualize its cellular distribution

  • Creating domain deletion mutants (e.g., GRAMD1CΔGRAM-eGFP) to study domain-specific localization patterns

  • TIRF microscopy to specifically examine recruitment to the plasma membrane

• Subcellular fractionation and biochemical approaches:

  • Isolation of mitochondria followed by western blotting to detect GRAMD1C association

  • Application of mCherry-tagged cholesterol binding domain of Perfringolysin O (mCherry-D4) to quantify mitochondrial cholesterol levels

• Dynamic translocation assays:

  • Treatment with sphingomyelinase to induce changes in accessible plasma membrane cholesterol and monitor GRAMD1C recruitment

  • Using methyl-β-cyclodextrin (MBCD) for cholesterol depletion experiments

These methodologies can be combined to provide comprehensive insights into GRAMD1C's dynamic behavior under various cellular conditions.

What are the best approaches for generating and validating GRAMD1C knockout models?

The literature documents several effective strategies for generating and validating GRAMD1C knockout models:

• Generation methods:

  • CRISPR/Cas9-mediated genome editing to create global GRAMD1C knockout mice (Gramd1c-/-)

  • siRNA or shRNA-mediated knockdown for cell culture studies

  • Generation of cell lines with GRAMD1C triple knockout (alongside other GRAMD1 family members)

• Validation approaches:

  • Genotyping using PCR with specific primers targeting the modified locus

  • Western blot analysis to confirm protein depletion

  • RT-qPCR to verify reduction of mRNA expression

  • Functional assays measuring cholesterol transport efficiency

• Phenotypic characterization:

  • Analysis of whole-body cholesterol balance in knockout mice fed different cholesterol diets

  • Assessment of sterol-derived metabolites in feces, liver, and plasma

  • Examination of mitochondrial function parameters (respiratory capacity, membrane potential)

Developing complementary in vitro and in vivo models provides robust validation of GRAMD1C function across different biological contexts.

How does GRAMD1C sense and transport accessible plasma membrane cholesterol?

GRAMD1C employs a sophisticated mechanism to sense and transport accessible plasma membrane cholesterol:

• Sensing mechanism:

  • The GRAM domain functions as a coincidence detector that recognizes both accessible cholesterol and anionic lipids including phosphatidylserine (PS)

  • These recognition events occur through distinct but synergistic binding sites within the GRAM domain

  • The GRAM domain facilitates cholesterol-dependent recruitment of GRAMD1C to membrane contact sites when accessible cholesterol levels exceed a certain threshold

• Transport process:

  • Once recruited, GRAMD1C extracts accessible cholesterol via its StART-like domain

  • This domain can directly capture and transport sterol across the cytoplasm

  • GRAMD1C then delivers this cholesterol to the ER, helping maintain cellular cholesterol homeostasis

• Regulatory features:

  • GRAMD1C's luminal helices are important for proper localization and function

  • Mutations in the hydrophobic residues of these helices disrupt the protein's ability to form patches on tubular ER, potentially affecting its transport capacity

This multi-step process allows cells to monitor and respond to fluctuations in accessible plasma membrane cholesterol levels, preventing potentially harmful accumulation.

What experimental evidence supports GRAMD1C's role in mitochondrial cholesterol regulation?

Multiple lines of experimental evidence support GRAMD1C's involvement in mitochondrial cholesterol regulation:

• Protein interaction data:

  • Proteomic analysis revealed that GRAMD1C primarily interacts with proteins of mitochondrial origin

  • These include mitochondrial proteins like NDUFAF2, SHDB, and ATAD3A, as well as ER-mitochondria contact site proteins VDAC1 and ACSL4

  • These interactions were absent when using GRAMD1C lacking the GRAM domain (ΔGRAM), indicating that mitochondrial interaction depends on this domain

• Mitochondrial cholesterol measurements:

  • Development of a quantification method using mCherry-tagged cholesterol binding domain of Perfringolysin O (mCherry-D4) applied to isolated mitochondria

  • MBCD treatment decreased mCherry-D4 binding, validating the assay's specificity for cholesterol

• Functional consequences of GRAMD1C depletion:

  • GRAMD1C knockdown resulted in increased mitochondrial cholesterol accumulation

  • Enhanced mitochondrial respiration and oxygen consumption rate were observed in GRAMD1C-depleted cells

  • These effects were not due to changes in mitochondrial proteome, membrane potential, or ROS production

Together, these findings establish GRAMD1C as a negative regulator of mitochondrial cholesterol abundance and mitochondrial bioenergetics.

What is known about GRAMD1C's role in cancer, particularly clear cell renal carcinoma (ccRCC)?

GRAMD1C has emerging significance in cancer biology, particularly in clear cell renal carcinoma (ccRCC):

These findings suggest that GRAMD1C may serve as a prognostic marker in ccRCC and potentially other cancers where lipid metabolism is dysregulated.

Are there known mutations in GRAMD1C associated with human diseases?

Yes, specific mutations in GRAMD1C have been associated with human disease:

• R189W mutation and intellectual disability:

  • A mutation within the GRAM domain of GRAMD1b (R189W) has been associated with intellectual disability in humans

  • Functional characterization revealed that this mutation specifically impairs cholesterol sensing without affecting the protein's affinity toward phosphatidylserine (PS)

  • Cell-free reconstitution assays demonstrated that GRAMD1b proteins with the R189W mutation:

    • Failed to tether membranes effectively

    • Transported cholesterol less efficiently compared to wild-type GRAMD1b proteins

• Functional consequences of mutations:

  • Re-expression of wild-type GRAMD1b, but not the R189W mutant, in GRAMD1 triple knockout cells restored proper regulation of SREBP-2

  • The wild-type protein also suppressed abnormal accumulation of accessible plasma membrane cholesterol, while the mutant could not

• Other functionally significant residues:

  • A screening approach identified glycine residue at position 187 (G187) as critical for the sensitivity of the GRAM domain to accessible plasma membrane cholesterol

  • Converting this residue to the more hydrophobic leucine (G187L) enhanced cholesterol sensitivity without altering PS affinity

  • This modification improved GRAMD1b-dependent membrane tethering and cholesterol transport

These findings highlight the importance of specific residues within the GRAM domain for proper cholesterol sensing and transport functions.

How does GRAMD1C interact with other GRAM domain family proteins?

GRAMD1C forms complex interactions with other GRAM domain family proteins:

These interactions likely allow for fine-tuned regulation of cholesterol transport and lipid homeostasis across different cellular compartments and physiological conditions.

What is the relationship between GRAMD1C and autophagy regulation?

GRAMD1C has been identified as a negative regulator of starvation-induced autophagy , though the detailed mechanisms remain to be fully elucidated:

• Experimental evidence:

  • Studies have described GRAMD1C as a negative regulator of starvation-induced autophagy

  • This function may be linked to its role in cholesterol transport between organelles

• Potential mechanisms:

  • GRAMD1C's regulation of mitochondrial cholesterol levels may influence mitochondrial function and subsequently affect autophagy

  • As autophagy is a key process in cellular stress response, GRAMD1C may connect lipid homeostasis to autophagy regulation

  • The protein's interactions with mitochondrial proteins could influence mitochondrial quality control pathways that intersect with autophagy

• Research approaches to investigate this relationship:

  • Monitoring autophagy markers (LC3-II, p62) in GRAMD1C knockout or overexpression models

  • Analyzing autophagic flux using tandem fluorescent-tagged LC3 reporters in cells with altered GRAMD1C levels

  • Examining selective autophagy pathways (e.g., mitophagy) given GRAMD1C's interaction with mitochondria

Further research is needed to fully understand the molecular mechanisms connecting GRAMD1C to autophagy regulation and how this relationship impacts cellular homeostasis under various stress conditions.

What are the most promising research directions for understanding GRAMD1C's physiological significance?

Several promising research directions could enhance our understanding of GRAMD1C's physiological significance:

• Tissue-specific functions:

  • While whole-body knockout models have been studied , investigating tissue-specific knockout models could reveal specialized functions

  • Particular focus on tissues with high cholesterol turnover (adrenal gland, brain, liver) would be valuable

• Interaction with other lipid transport systems:

  • Exploring potential crosstalk between GRAMD1C and other cholesterol transport proteins

  • Investigating compensatory mechanisms in GRAMD1C-deficient models

• Role in specific disease contexts:

  • Expanding studies beyond ccRCC to other cancers with dysregulated lipid metabolism

  • Investigating potential roles in neurodegenerative diseases, given the R189W mutation's association with intellectual disability

• Therapeutic potential:

  • Assessing whether modulation of GRAMD1C activity could normalize cholesterol homeostasis in disease states

  • Developing small molecule regulators that could enhance or inhibit specific GRAMD1C functions

These research directions would significantly advance our understanding of GRAMD1C's role in health and disease.

What methodological innovations could advance GRAMD1C research?

Emerging technologies and methodological innovations could substantially advance GRAMD1C research:

• Advanced imaging approaches:

  • Super-resolution microscopy to better visualize GRAMD1C at membrane contact sites

  • Live-cell imaging with genetically encoded cholesterol sensors to track GRAMD1C-mediated cholesterol movement in real-time

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography studies of GRAMD1C alone and in complex with lipids

  • Structural analysis of the conformational changes that occur during cholesterol binding and transport

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and lipidomics in GRAMD1C models

  • Network analysis to position GRAMD1C within broader lipid homeostasis pathways

• Precision gene editing:

  • CRISPR-based approaches to introduce specific mutations (like R189W or G187L) to create disease models

  • Base editing to create specific point mutations with minimal off-target effects

These methodological innovations would provide deeper insights into GRAMD1C's molecular mechanisms and physiological functions.