CHCHD3 Human

What is CHCHD3 and what is its primary function in human cells?

CHCHD3 (also known as MIC19) is an inner mitochondrial membrane scaffold protein that serves as a key component of the mitochondrial contact site and cristae organizing system (MICOS). This protein plays crucial roles in maintaining crista junctions, inner membrane architecture, and formation of contact sites to the outer membrane. It functions as a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import, making it essential for mitochondrial structure and function .

Beyond its structural role, CHCHD3 also functions as a transcription factor that binds to the BAG1 promoter and represses BAG1 transcription . The absence of CHCHD3 affects the structural integrity of mitochondrial cristae and leads to significant reductions in ATP production, cell growth, and oxygen consumption .

What are the key structural features of human CHCHD3 protein?

Human CHCHD3 has several distinctive structural features that contribute to its function:

• N-terminal myristoylation: CHCHD3 has a well-conserved myristoylation site at the N-terminal Gly-2, which is essential for proper protein localization and biological function. Mass spectrometric analysis has confirmed that the N-terminal glycine is myristoylated .

• PKA phosphorylation site: The protein contains a PKA phosphorylation site at Thr-11, which may regulate its function .

• CHCH domains: As its name suggests, CHCHD3 contains coiled-coil-helix-coiled-coil-helix domains that are characteristic of this protein family and are critical for its interactions with other mitochondrial proteins .

• Mitochondrial localization: CHCHD3 resides primarily on the inner mitochondrial membrane facing the intermembrane space, with trace amounts associated with the outer membrane .

What protein complexes does CHCHD3 interact with in the mitochondria?

CHCHD3 interacts with several key proteins that are essential for mitochondrial structure and function:

• Mitofilin: An inner membrane protein that regulates crista morphology .

• OPA1: An inner membrane protein involved in mitochondrial fusion and crista structure maintenance .

• Sam50: An outer membrane protein that regulates import and assembly of β-barrel proteins on the outer membrane .

These interactions form part of a larger network that maintains mitochondrial architecture. Knockdown of CHCHD3 leads to almost complete loss of both mitofilin and Sam50 proteins and alterations in several other mitochondrial proteins, highlighting its role as a central scaffolding protein in maintaining mitochondrial structure .

What are the most effective methods for studying CHCHD3 localization in human cells?

Researchers have successfully employed multiple complementary techniques to accurately determine CHCHD3 localization:

• Submitochondrial fractionation: This method has been particularly effective for determining the precise location of CHCHD3 within mitochondria. By fractionating purified mitochondria into outer membrane, intermembrane space, inner membrane, and matrix compartments, researchers have shown that CHCHD3 is primarily enriched in the inner membrane fraction with trace amounts in the outer membrane .

• Immunofluorescence microscopy: Confocal microscopy using specific antibodies against CHCHD3 and mitochondrial markers provides visual confirmation of its mitochondrial localization .

• Electron microscopy: For ultra-structural studies, transmission electron microscopy has been invaluable in assessing the impact of CHCHD3 knockdown on crista morphology and mitochondrial structure .

• Mass spectrometry: This technique has been used to confirm post-translational modifications such as N-terminal myristoylation, which influences CHCHD3 localization and function .

What experimental approaches are recommended for studying CHCHD3 protein-protein interactions?

Several complementary techniques have proven effective for investigating CHCHD3 interactions:

• Co-immunoprecipitation (Co-IP): For CHCHD3 interaction studies, researchers have successfully immunoprecipitated CHCHD3 from gradient-purified mitochondria using specific antibodies. Pre-clearing samples on protein A-agarose beads prevents non-specific binding. After immunocapture with protein A-agarose beads, samples are analyzed by SDS-PAGE followed by immunoblotting for potential interaction partners .

• Cross-linking followed by mass spectrometry: This approach can identify transient or weak interactions that might be missed by Co-IP alone.

• Proximity labeling techniques (BioID or APEX): These methods can identify proteins in close proximity to CHCHD3 in living cells, providing a more physiological context for interaction studies.

• Yeast two-hybrid screening: While not mentioned specifically in the search results, this is a standard approach that could identify novel CHCHD3 interaction partners.

• Genetic interaction screens: Studies in Drosophila have revealed genes that genetically interact with the CHCHD3 homolog (Chchd3/6), including Cdk12, RNF149, and SPTBN1 .

What are reliable methods for knockdown or knockout of CHCHD3 in experimental systems?

Researchers have successfully employed several approaches to modulate CHCHD3 expression:

• RNA interference (RNAi): RNAi knockdown in HeLa cells has been effectively used to study the consequences of CHCHD3 depletion. This approach revealed that CHCHD3 knockdown results in fragmented mitochondria, reduced OPA1 protein levels, impaired fusion, and clustering of mitochondria around the nucleus .

• Tissue-specific knockdown in model organisms: In Drosophila, tissue-specific RNAi knockdown using the GAL4-UAS system has been employed to study the temporal and spatial requirements of Chchd3/6 (the Drosophila homolog). Knockdown was achieved using multiple GAL4 drivers including Hand4.2-Gal4 (cardiac-specific), Dot-Gal4 (pericardial cells), Mef2-Gal4 (pan-muscle), and elav-Gal4 (pan-neuronal) .

• CRISPR-Cas9 genome editing: While not explicitly mentioned in the search results, this is now a standard approach for generating knockout cell lines and organisms.

• Verification strategies: When performing knockdown experiments, it's crucial to verify specificity. For example, in Drosophila studies, researchers confirmed that cardiac effects were specifically due to Chchd3/6 knockdown by testing a predicted off-target gene (Duox) and demonstrating that its knockdown had no effect on heart function .

How does CHCHD3 dysfunction contribute to mitochondrial diseases?

CHCHD3 dysfunction significantly impairs mitochondrial structure and function, potentially contributing to mitochondrial disease through several mechanisms:

• Disruption of crista architecture: RNAi knockdown of CHCHD3 in HeLa cells results in aberrant mitochondrial inner membrane structures with fragmented and tubular cristae or complete loss of cristae, and reduced crista membrane. The crista junction opening diameter is reduced to 50%, suggesting significant remodeling of cristae in the absence of CHCHD3 .

• Impaired bioenergetics: Both oxygen consumption and glycolytic rates are severely restricted in CHCHD3-depleted cells, indicating compromised mitochondrial function .

• Altered mitochondrial dynamics: CHCHD3 knockdown results in fragmented mitochondria, reduced OPA1 protein levels, and impaired fusion capability. Additionally, the mitochondria cluster around the nucleus, and cellular growth rate is reduced .

• Disruption of protein complexes: Loss of CHCHD3 leads to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, destabilizing critical mitochondrial protein complexes .

• Disease associations: CHCHD3 has been linked to conditions such as Barth Syndrome and was found to be significantly down-regulated in a cell line model of familial amyotrophic lateral sclerosis expressing SOD1 mutant G93A .

What evidence links CHCHD3 to cardiac disorders, particularly hypoplastic left heart syndrome (HLHS)?

Recent research has established connections between CHCHD3 and cardiac disorders:

• Human genetic studies: Whole genome sequencing of a family with hypoplastic left heart syndrome (HLHS) identified CHCHD3 as a potential contributor to the condition. In one studied family (11H), a homozygous recessive disease mode of inheritance was proposed due to reported consanguinity between the mother and father .

• Latent cardiac dysfunction: The HLHS proband with CHCHD3 mutations showed latent decline of right ventricular function, suggesting that CHCHD3 may contribute not only to congenital heart defects but also to later-onset cardiac dysfunction .

• Drosophila model evidence: Knockdown of Chchd3/6 (the Drosophila homolog) resulted in decreased heart tube contractility and systolic dysfunction in fly models. This phenotype was observed in both intact flies and in semi-intact adult heart preparations lacking neuronal inputs .

• Cardiomyocyte-autonomous effects: Tissue-specific knockdown experiments in Drosophila demonstrated that cardiac dysfunction occurs with muscle-specific knockdown but not with pericardial cell or neuronal knockdown, confirming cardiomyocyte-autonomous effects of Chchd3/6 .

• Developmental and maintenance roles: Expression analysis showed that Chchd3/6 is expressed in Drosophila cardioblasts during embryonic development. Additionally, research suggests that Chchd3/6 is required both during pupal development and at adult stages for maintaining robust heart function .

What is known about CHCHD3 mutations in human disease?

While the available search results provide limited information on specific CHCHD3 mutations, they do offer some insights:

• Disease associations: CHCHD3 has been associated with Barth Syndrome and potentially with hypoplastic left heart syndrome (HLHS) .

• Mutation database: According to the information provided, there are 8 recorded mutations in the CHCHD3 gene , although specific details about these mutations are not provided in the search results.

• Functional consequences: The search results suggest that mutations affecting CHCHD3 function could impact mitochondrial cristae structure, ATP production, cell growth, and oxygen consumption .

• Familial cases: In at least one family with HLHS, a homozygous recessive inheritance pattern involving CHCHD3 was proposed, suggesting that recessive mutations in this gene may contribute to congenital heart defects .

• Research models: Studies in Drosophila have provided evidence that disruption of the CHCHD3 homolog (Chchd3/6) leads to cardiac dysfunction, supporting the potential pathogenicity of CHCHD3 mutations in human cardiac disease .

How does CHCHD3 coordinate with other MICOS complex components to maintain mitochondrial cristae structure?

CHCHD3 (MIC19) functions as a central component within the MICOS complex through multiple coordinated interactions:

• Scaffolding function: CHCHD3 serves as a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture. It interacts with other MICOS components to form a functional complex that maintains the structural integrity of cristae junctions .

• Interaction with mitofilin: CHCHD3 interacts directly with mitofilin (another key component of MICOS), and knockdown of CHCHD3 leads to almost complete loss of mitofilin, suggesting that CHCHD3 is essential for mitofilin stability .

• OPA1 regulation: CHCHD3 interacts with OPA1, a dynamin-like GTPase involved in inner membrane fusion and cristae remodeling. RNAi knockdown of CHCHD3 results in reduced OPA1 protein levels, suggesting that CHCHD3 may regulate OPA1 stability or expression .

• Cross-membrane coordination: CHCHD3 not only interacts with inner membrane proteins but also with the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins. This suggests that CHCHD3 plays a role in coordinating the structure and function of both mitochondrial membranes .

• Cristae junction regulation: Ultrastructural analysis of cells with CHCHD3 knockdown revealed that the crista junction opening diameter was reduced to 50%, suggesting that CHCHD3 plays a specific role in regulating the architecture of these critical structures .

What is the relationship between CHCHD3's role as a transcription factor and its mitochondrial functions?

CHCHD3 has a dual role as both a mitochondrial structural protein and a transcription factor, raising intriguing questions about the coordination of these functions:

• Transcriptional repression: CHCHD3 has been shown to function as a transcription factor which binds to the BAG1 promoter and represses BAG1 transcription . BAG1 (BCL2 Associated Athanogene 1) is a regulator of apoptosis and cellular stress responses.

• Compartmentalization question: The search results don't explicitly address how CHCHD3 can function both within mitochondria and as a nuclear transcription factor. This raises questions about whether different pools of the protein exist in different cellular compartments, or whether it shuttles between compartments under specific conditions.

• Potential signaling role: The dual localization and function suggest that CHCHD3 might play a role in mitochondria-to-nucleus signaling, potentially linking mitochondrial status to nuclear gene expression.

• Regulatory modifications: The presence of a PKA phosphorylation site on CHCHD3 suggests potential regulation by signaling pathways, which might influence its localization or function as either a mitochondrial protein or a transcription factor .

• Evolutionary significance: The dual function may represent an evolutionary adaptation that allows coordinated regulation of mitochondrial structure and nuclear gene expression in response to cellular demands.

What is the temporal requirement of CHCHD3 expression during development and adult tissue maintenance?

Research, particularly in Drosophila models, has provided insights into the temporal requirements of CHCHD3:

• Embryonic expression: Analysis of single-cell transcriptomic data from Drosophila embryos showed that Chchd3/6 (the Drosophila homolog) is expressed in cardioblasts during embryonic development, along with other cardiogenic factors like tinman, H15, and Hand .

• Developmental requirement: Studies in Drosophila suggest that Chchd3/6 plays an important role during heart development, as evidenced by its expression in developing cardioblasts .

• Adult tissue maintenance: Research has indicated that "Chchd3/6 is not only required during pupal development, but also at adult stages for maintaining robust heart function" . This suggests an ongoing requirement for CHCHD3 in maintaining tissue function beyond development.

• Temporal-specific knockdown: While not explicitly detailed in the search results, temporal-specific knockdown experiments (perhaps using inducible systems) would be valuable to further determine the precise temporal requirements for CHCHD3 at different developmental stages and in adult tissues.

• Progressive phenotypes: In the HLHS proband with CHCHD3 mutations, there was a "latent decline of right ventricular function" , suggesting that CHCHD3 dysfunction may lead to progressive deterioration of tissue function over time.

What are the most effective approaches for studying CHCHD3's role in mitochondrial dynamics?

Researchers have employed several sophisticated methodologies to investigate CHCHD3's impact on mitochondrial dynamics:

• Live-cell imaging: Visualization of mitochondrial network morphology and dynamics in real-time using fluorescent proteins targeted to mitochondria in cells with normal or altered CHCHD3 expression.

• Fluorescence recovery after photobleaching (FRAP): This technique can assess the mobility and dynamics of CHCHD3 and its interaction partners within mitochondrial compartments.

• RNAi and phenotypic analysis: RNAi knockdown of CHCHD3 in HeLa cells revealed its importance in mitochondrial dynamics, as evidenced by fragmented mitochondria, reduced OPA1 protein levels, impaired fusion, and clustering of mitochondria around the nucleus .

• Transmission electron microscopy: This technique provides high-resolution images of mitochondrial ultrastructure, allowing assessment of cristae morphology in cells with normal or altered CHCHD3 expression. Studies have shown that CHCHD3 knockdown results in aberrant mitochondrial inner membrane structures with fragmented and tubular cristae or loss of cristae .

• Functional assays: Measurements of oxygen consumption, ATP production, and glycolytic rates provide insights into the functional consequences of altered mitochondrial dynamics due to CHCHD3 manipulation .

How can researchers effectively model CHCHD3-associated diseases in experimental systems?

Several experimental models have proven valuable for investigating CHCHD3-associated diseases:

• Cell culture models:

  • RNAi knockdown in human cell lines (e.g., HeLa cells) has been effective for studying cellular consequences of CHCHD3 deficiency

  • CRISPR-Cas9-mediated knockout or mutation of CHCHD3 can create stable cellular models

• Drosophila models:

  • Tissue-specific knockdown using the GAL4-UAS system has been successfully employed to study cardiac-specific effects of Chchd3/6 deficiency

  • Both intact fly cardiac assays and semi-intact adult heart preparations (SOHA) have been used to assess heart function

• Disease-specific models:

  • For cardiac disorders such as HLHS, both Drosophila heart models and mammalian cardiac cell systems can be used

  • Patient-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes or other relevant cell types would be valuable for studying patient-specific mutations

• Temporal control systems:

  • Inducible knockdown or knockout systems allow investigation of temporal requirements for CHCHD3 during development and in adult tissues

  • This approach has revealed that Chchd3/6 is required both during pupal development and at adult stages for maintaining robust heart function

• Validation strategies:

  • When modeling disease-associated mutations, it's important to validate findings across multiple model systems

  • For RNAi studies, testing for off-target effects is crucial, as demonstrated in Drosophila studies where potential off-target effects were systematically ruled out

What are the key unanswered questions regarding CHCHD3 function and disease associations?

Several critical questions remain to be addressed in CHCHD3 research:

• Structure-function relationships: How do specific domains and post-translational modifications of CHCHD3 contribute to its various functions in mitochondrial structure maintenance and transcriptional regulation?

• Regulatory mechanisms: What signaling pathways and cellular conditions regulate CHCHD3 expression, localization, and function? How does the PKA phosphorylation site at Thr-11 impact CHCHD3 function?

• Disease mechanisms: How do specific CHCHD3 mutations lead to diseases such as Barth Syndrome or contribute to hypoplastic left heart syndrome? Are there other diseases associated with CHCHD3 dysfunction that remain to be identified?

• Dual localization: How does CHCHD3 function both as a mitochondrial protein and as a nuclear transcription factor? Does it shuttle between compartments, and if so, what regulates this shuttling?

• Therapeutic potential: Could targeting CHCHD3 or its downstream pathways provide therapeutic benefits for mitochondrial diseases or cardiac disorders associated with CHCHD3 dysfunction?

What emerging technologies might advance our understanding of CHCHD3 biology?

Several cutting-edge technologies hold promise for deepening our understanding of CHCHD3:

• Cryo-electron microscopy: High-resolution structural studies of CHCHD3 and its complexes within the mitochondrial membrane would provide valuable insights into its molecular function.

• Single-cell multi-omics: Integrating transcriptomics, proteomics, and metabolomics at the single-cell level could reveal cell-type-specific roles of CHCHD3 and how its dysfunction affects cellular processes differently across tissues.

• Live-cell super-resolution microscopy: These techniques could provide unprecedented visualization of CHCHD3's dynamic behavior and interactions within mitochondria.

• Mitochondrial-specific proximity labeling: Techniques such as mito-APEX or split-BioID could identify the complete interactome of CHCHD3 within the mitochondrial compartment.

• In situ structural biology: Emerging techniques that allow visualization of protein structures within cells could reveal how CHCHD3 is organized within the context of intact mitochondria.

How might targeting CHCHD3 or its pathways lead to therapeutic approaches for mitochondrial disorders?

Several therapeutic strategies targeting CHCHD3 pathways could be explored:

• Gene therapy approaches: For loss-of-function mutations, delivery of functional CHCHD3 to affected tissues could potentially restore mitochondrial structure and function.

• Small molecule modulators: Compounds that stabilize CHCHD3 protein or promote its proper interactions with partners like mitofilin, OPA1, and Sam50 could help maintain mitochondrial cristae structure.

• Bypass strategies: Identifying and targeting downstream effectors of CHCHD3 might provide alternative approaches to restore mitochondrial function in cases of CHCHD3 dysfunction.

• Metabolic interventions: Since CHCHD3 dysfunction affects mitochondrial energy production, metabolic interventions that bypass affected pathways could potentially alleviate symptoms.

• Early intervention in cardiac disease: Given the evidence linking CHCHD3 to cardiac disorders like HLHS and the observation that the HLHS proband showed latent decline of ventricular function, early interventions targeting CHCHD3 pathways might prevent or delay cardiac deterioration in susceptible individuals .

What are the recommended research tools and resources for studying CHCHD3?

Researchers interested in CHCHD3 can utilize various tools and resources:

What are the key CHCHD3 research publications that established foundational knowledge?

While the search results don't provide a comprehensive publication history, they reference several important studies:

• Studies characterizing CHCHD3 as a PKA substrate (Schauble et al., 2007)

• Research identifying CHCHD3 as a component of the MICOS complex

• Work demonstrating CHCHD3's role in maintaining crista integrity and mitochondrial function

• Studies showing CHCHD3's interaction with mitofilin, OPA1, and Sam50

• Research linking CHCHD3 to hypoplastic left heart syndrome (HLHS)

• Work establishing CHCHD3's dual role as both a mitochondrial structural protein and a transcription factor

• Studies in Drosophila revealing genetic interactions between Chchd3/6 and genes like Cdk12, RNF149, and SPTBN1