Recombinant Bovine Coiled-coil domain-containing protein 47 (CCDC47)

What is CCDC47 and what are its key structural features?

CCDC47 (Coiled-coil domain-containing protein 47), also known as calumin, is an ER transmembrane calcium-binding protein widely expressed in mammalian tissues including brain, lung, heart, stomach, liver, spleen, kidney, muscle, and testis . Structurally, human CCDC47 contains an N-terminal signal peptide and a single transmembrane helix . The protein possesses coiled-coil domains that likely contribute to its functional interactions with other proteins. Structural modeling studies using RaptorX-Contact have generated models of the human protein that provide insights into its potential three-dimensional conformation .

What is the biological function of CCDC47?

CCDC47 functions as a calcium-binding protein in the ER with low affinity but high capacity for calcium ions. It plays critical roles in calcium signaling and is essential for embryonic development . Recent research has identified CCDC47 as part of a specialized ER translocon complex involved in the biogenesis of multi-pass membrane proteins . While still being fully characterized, CCDC47 appears to be involved in various membrane-associated processes and has been linked to calcium homeostasis in the ER, which is vital for numerous cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation .

How conserved is CCDC47 across species?

While specific conservation data for bovine CCDC47 isn't directly provided in the search results, CCDC47 appears to be highly conserved across mammalian species. Conservation analysis using ConSurf scores shows varying degrees of conservation throughout the human CCDC47 protein sequence . The functional importance of CCDC47 is highlighted by the fact that knockout of Ccdc47 in mice causes embryonic lethality, suggesting that its critical functions are likely conserved across mammalian species including bovine .

What are the optimal storage conditions for recombinant CCDC47?

Recombinant CCDC47 is typically provided in liquid form containing glycerol. For optimal stability, it should be stored at -20°C, and for extended storage, it's recommended to conserve the protein at -20°C or -80°C . Repeated freezing and thawing should be avoided to maintain protein integrity. For short-term use (up to one week), working aliquots can be stored at 4°C .

How can I validate the identity and activity of recombinant bovine CCDC47?

Validation of recombinant bovine CCDC47 can be performed through several complementary approaches:

• Western blotting: Using specific antibodies against CCDC47 to confirm protein identity and molecular weight

• Mass spectrometry: For precise protein identification and detection of potential modifications

• Calcium binding assays: To verify the calcium-binding capacity of the recombinant protein

• Functional assays: Evaluating the protein's ability to influence calcium signaling in appropriate cellular models

These methods can be combined to ensure both the structural and functional integrity of the recombinant protein before use in experimental applications.

How can recombinant CCDC47 be used to study calcium homeostasis in the ER?

Recombinant CCDC47 can serve as a valuable tool for investigating calcium homeostasis in the ER through various experimental approaches:

• Reconstitution experiments: Introducing recombinant CCDC47 into model membrane systems to study its direct effects on calcium handling

• Rescue experiments: Using recombinant CCDC47 to complement CCDC47-deficient cells or tissues to restore calcium signaling functions

• Interaction studies: Identifying binding partners that connect CCDC47 to the calcium signaling machinery

• Structure-function analysis: Creating modified versions of CCDC47 to determine which domains are essential for calcium binding and signaling

Cellular studies have shown that CCDC47 deficiency leads to decreased total ER calcium storage, impaired calcium signaling mediated by the IP3R calcium release channel, and reduced ER calcium refilling via store-operated calcium entry (SOCE) .

What cellular assays are appropriate for studying CCDC47's role in calcium signaling?

Several cellular assays can be employed to investigate CCDC47's involvement in calcium signaling:

• Calcium imaging: Using fluorescent calcium indicators (e.g., Fura-2, Fluo-4) to monitor changes in cytosolic and ER calcium levels

• Patch-clamp electrophysiology: Measuring calcium currents through store-operated calcium channels

• FRET-based sensors: Employing genetically encoded calcium indicators to monitor calcium dynamics in specific cellular compartments

• Store-operated calcium entry (SOCE) assays: Evaluating the impact of CCDC47 manipulation on SOCE by measuring calcium influx following ER calcium store depletion

• ER calcium measurement: Using ER-targeted calcium sensors to directly assess ER calcium content

These assays can be performed in cellular models with normal, reduced, or enhanced CCDC47 expression to determine the protein's specific contributions to calcium homeostasis.

How does CCDC47 contribute to embryonic development?

CCDC47 plays a critical role in embryonic development, as evidenced by studies in a Ccdc47-knockout mouse model. These mice exhibited:

• Delayed development

• Atrophic neural tubes

• Heart abnormalities

• Paucity of blood cells in the dorsal aorta

• Embryonic lethality

The essential nature of CCDC47 in development is likely related to its role in calcium signaling, as proper calcium homeostasis is required for numerous developmental processes including cell division, differentiation, and morphogenesis.

What human disorders are associated with CCDC47 dysfunction?

Bi-allelic variants in CCDC47 have been identified in individuals with a complex multisystem disorder characterized by:

• Woolly hair

• Liver dysfunction

• Pruritus

• Dysmorphic features

• Hypotonia

• Global developmental delay

Cellular studies from affected individuals showed decreased CCDC47 mRNA expression and protein levels, along with impaired calcium signaling. This suggests that dysregulation of calcium homeostasis due to CCDC47 dysfunction underlies the pathogenesis of this disorder .

What experimental models are available for studying CCDC47-related diseases?

Based on current research, several experimental models can be utilized to study CCDC47-related diseases:

• Patient-derived cells: Primary cells or cell lines derived from individuals with CCDC47 mutations

• Mouse models: While complete knockout is embryonically lethal, conditional or tissue-specific knockout models could be developed

• CRISPR-Cas9 engineered cell lines: Introducing specific disease-associated mutations into cellular models

• iPSC models: Generating induced pluripotent stem cells from patient samples and differentiating them into relevant cell types

These models provide complementary approaches to understand how CCDC47 mutations affect cellular function and contribute to disease phenotypes.

How does CCDC47 interact with the ER translocon machinery?

Recent research has identified CCDC47 as a component of a specialized ER translocon involved in multi-pass membrane protein biogenesis . This complex includes TMCO1, NICALIN, TMEM147, and NOMO. CCDC47 can be stably isolated with TMCO1-bound ribosome-Sec61 complexes, suggesting its involvement in co-translational processes at the ER . Understanding the precise interactions between CCDC47 and other translocon components requires advanced biochemical and structural approaches, including:

• Co-immunoprecipitation: To identify direct binding partners

• Proximity labeling: To map the spatial relationships between translocon components

• Cryo-electron microscopy: To determine the structural arrangement of the translocon complex

• Crosslinking mass spectrometry: To identify specific interaction interfaces

What techniques can be used to study the calcium-binding properties of CCDC47?

Several sophisticated techniques can be employed to characterize the calcium-binding properties of recombinant bovine CCDC47:

• Isothermal titration calorimetry (ITC): To measure binding affinities and thermodynamic parameters

• Surface plasmon resonance (SPR): For real-time analysis of calcium binding kinetics

• Circular dichroism (CD) spectroscopy: To monitor calcium-induced conformational changes

• Fluorescence spectroscopy: Using intrinsic tryptophan fluorescence or extrinsic fluorophores to detect calcium-dependent structural alterations

• NMR spectroscopy: To identify specific calcium-binding residues and characterize structural changes upon calcium binding

These approaches can provide detailed insights into how CCDC47 binds calcium, which is critical for understanding its role in calcium homeostasis.

How can I investigate CCDC47's role in the regulation of multi-pass membrane protein biogenesis?

To investigate CCDC47's involvement in multi-pass membrane protein biogenesis, researchers can implement these advanced approaches:

• Pulse-chase experiments: To track the synthesis, folding, and trafficking of model multi-pass membrane proteins in the presence or absence of functional CCDC47

• Ribosome profiling: To identify translational pauses that might be regulated by CCDC47 during membrane protein synthesis

• Proteomics analysis: To identify changes in the membrane proteome upon CCDC47 depletion or overexpression

• In vitro translation systems: Using reconstituted translation systems with purified components to directly assess CCDC47's role in membrane protein integration

• Single-molecule FRET: To visualize the dynamics of membrane protein insertion and folding in real-time

These methodologies can elucidate the molecular mechanisms by which CCDC47 contributes to the proper biogenesis of complex membrane proteins.

What are the current gaps in our understanding of CCDC47 function?

Despite progress in characterizing CCDC47, several aspects of its biology remain incompletely understood:

• Precise calcium-binding mechanism: The structural basis for calcium binding and its impact on CCDC47 conformation

• Species-specific differences: How bovine CCDC47 might differ functionally from human or rodent homologs

• Regulation of CCDC47: Factors controlling CCDC47 expression, localization, and activity

• Substrate specificity: Which specific membrane proteins depend on CCDC47 for proper biogenesis

• Tissue-specific functions: How CCDC47's role might vary across different tissues and cell types

What emerging technologies could advance CCDC47 research?

Future research on bovine CCDC47 could benefit from several cutting-edge technologies:

• AlphaFold/RoseTTAFold: For improved protein structure prediction

• Single-cell proteomics: To examine cell-to-cell variation in CCDC47 function

• Spatial transcriptomics and proteomics: To map CCDC47 distribution and interactions within tissues

• Organoid models: To study CCDC47 function in more physiologically relevant 3D cellular systems

• CRISPR screening: To identify genetic modifiers of CCDC47 function

These approaches could provide new insights into CCDC47 biology and potentially reveal novel therapeutic strategies for CCDC47-related disorders.

How might research on CCDC47 contribute to therapeutic developments?

Understanding CCDC47's role in calcium homeostasis and membrane protein biogenesis could lead to several therapeutic applications:

• Targeting calcium signaling disorders: Developing interventions that modulate CCDC47 function to restore normal calcium homeostasis

• Enhancing membrane protein biogenesis: Using insights from CCDC47 research to improve the production and folding of challenging membrane proteins, including drug targets

• Treating CCDC47-related disorders: Developing personalized approaches for individuals with CCDC47 mutations

• Biomarker development: Utilizing CCDC47 as a potential biomarker for disorders involving ER dysfunction or calcium dysregulation