Recombinant Human Putative coiled-coil-helix-coiled-coil-helix domain-containing protein CHCHD2P9, mitochondrial (CHCHD2P9)

What is known about the structural characterization of CHCH domain proteins?

The CHCH domain represents a defining structural feature of proteins like CHCHD2 and potentially CHCHD2P9. This domain is characterized by twin CX9C motifs that form a specific coiled-coil-helix-coiled-coil-helix structure stabilized by disulfide bonds between cysteine residues . The four cysteine residues form two disulfide bonds that play a critical role in stabilizing the helix-turn-helix fold of the domain. These structural features are essential for protein function and mitochondrial import processes.

CHCH domain-containing proteins are imported to the mitochondrial intermembrane space (IMS) through the disulfide relay-dependent Mia40 machinery . The formation of disulfide bonds within the CHCH domain is crucial for proper protein folding and stability within the oxidative environment of the mitochondrial intermembrane space. Research has shown that disruption of these disulfide bonds can lead to protein instability and potentially contribute to disease processes, as seen with certain mutations in CHCHD2 and CHCHD10. While detailed structural information specific to CHCHD2P9 is limited, researchers can use the structural characteristics of related CHCH domain proteins as a starting point for investigations.

What is the predicted subcellular localization of CHCHD2P9?

Based on its similarity to CHCHD2, CHCHD2P9 would theoretically localize to the mitochondrial intermembrane space (IMS) if expressed as a functional protein. The parent protein CHCHD2 has been definitively shown to localize primarily to the mitochondria, specifically in the mitochondrial intermembrane space . This localization is facilitated by the characteristic CHCH domain and its interaction with the mitochondrial import machinery. In response to cellular stress, CHCHD2 can translocate from the mitochondria to the nucleus, where it functions as a transcription factor .

Interestingly, immunofluorescence studies of CHCHD2 have detected its presence in both the endoplasmic reticulum and mitochondria, suggesting a potential role in the communication between these organelles . This dual localization might represent an important aspect of CHCH domain-containing proteins in coordinating cellular responses to stress. If CHCHD2P9 produces a functional protein, similar localization patterns might be expected, though this would require experimental verification using specific antibodies or tagged recombinant proteins to accurately determine its subcellular distribution.

How is CHCHD2P9 expression regulated in different tissues?

While specific information about CHCHD2P9 expression patterns is limited in the current literature, insights can be drawn from what is known about CHCHD2 regulation. CHCHD2 shows differential expression across tissues, with particularly notable expression in tissues with high energy demands . Understanding whether CHCHD2P9 follows similar tissue-specific expression patterns would provide valuable insights into its potential biological roles.

CHCHD2 expression has been shown to be regulated by hypoxic conditions, with the protein functioning as a transcription factor that binds to oxygen responsive elements under both hypoxic (4% oxygen) and normoxic (20% oxygen) conditions . This oxygen-responsive regulation suggests an important role in cellular adaptation to varying oxygen levels. Additionally, CHCHD2 expression is significantly altered in various cancer types, with studies showing upregulation in hepatocellular carcinoma, breast cancer, and renal cell carcinoma . Whether CHCHD2P9 exhibits similar regulatory responses to metabolic stress or is differentially expressed in disease states remains an important question for future research.

What protein-protein interactions might be predicted for CHCHD2P9?

Based on the established interactions of CHCHD2, several potential protein-protein interactions might be predicted for CHCHD2P9 if it produces a functional protein. CHCHD2 has been shown to interact with BCL-XL, playing a crucial role in inhibiting mitochondria-mediated apoptosis by preventing the mitochondrial accumulation and oligomerization of Bax . This interaction represents an important mechanism by which CHCHD2 regulates cell survival under stress conditions.

CHCHD2 also interacts with components of the mitochondrial respiratory chain, particularly with cytochrome c, influencing mitochondrial crista structure and oxidative phosphorylation efficiency . The loss of CHCHD2 has been shown to affect mitochondrial crista structure and destabilize cytochrome c, highlighting its importance in maintaining mitochondrial integrity . Additionally, CHCHD2 has been found to interact with the recombination signal sequence-binding protein J in the nucleus, where they cooperatively regulate gene expression . Whether CHCHD2P9 retains any of these interaction capabilities would depend on the preservation of key structural features and would require specific biochemical and cellular assays to determine.

What are the recommended protocols for expressing and purifying recombinant CHCHD2P9?

Recombinant expression of CHCHD2P9 would typically follow standard molecular biology approaches used for other mitochondrial proteins. The coding sequence for CHCHD2P9 should be cloned into an appropriate expression vector with a purification tag such as His6, GST, or FLAG. For bacterial expression, E. coli strains capable of forming disulfide bonds (such as Origami or SHuffle) may be preferable given the importance of disulfide bonds in the CHCH domain structure . Alternatively, mammalian or insect cell expression systems might provide more appropriate post-translational modifications.

Purification would typically involve affinity chromatography based on the chosen tag, followed by size exclusion chromatography to ensure homogeneity. Special consideration should be given to the oxidative state of the protein during purification, as the formation of proper disulfide bonds within the CHCH domain is critical for stability and function. Some researchers have successfully used specialized buffers containing defined ratios of oxidized and reduced glutathione to promote proper disulfide bond formation during protein folding. Following purification, verification of proper folding using circular dichroism or other structural approaches would be advisable to ensure that the recombinant protein maintains the expected CHCH domain structure.

What analytical techniques are most suitable for studying CHCHD2P9?

Multiple complementary analytical techniques would be valuable for characterizing CHCHD2P9 structure and function. For structural analysis, circular dichroism (CD) spectroscopy can provide information about secondary structure content, while nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography would offer higher-resolution structural details. Mass spectrometry approaches, particularly hydrogen-deuterium exchange mass spectrometry (HDX-MS), could provide insights into protein dynamics and regions involved in protein-protein interactions.

For functional characterization, researchers might employ various biochemical assays depending on the hypothesized functions based on CHCHD2. These could include DNA binding assays if transcription factor activity is suspected, oxygen consumption measurements to assess effects on mitochondrial respiration, or apoptosis assays to investigate potential roles in cell death regulation . Protein-protein interaction studies using co-immunoprecipitation, proximity ligation assays, or fluorescence resonance energy transfer (FRET) could help identify binding partners. In cellular contexts, fluorescently tagged CHCHD2P9 constructs could enable visualization of subcellular localization and potential translocation under various stress conditions, similar to what has been observed with CHCHD2 .

What research models are available for studying CHCHD2P9?

Various research models could be employed to study CHCHD2P9, ranging from cellular systems to animal models. Cell culture models using cell lines with high mitochondrial activity (such as neuronal or muscle cell lines) would be valuable for investigating localization, expression patterns, and functional effects of CHCHD2P9 overexpression or knockdown. CRISPR-Cas9 genome editing could be used to create cell lines with modified CHCHD2P9 expression for more detailed functional studies.

Animal models have proven useful for studying related proteins like CHCHD2. Drosophila models have provided insights into the consequences of CHCHD2 loss, showing ROS-dependent apoptosis via destabilization of cytochrome c . Mouse models of CHCHD2 disruption have similarly revealed important phenotypes related to mitochondrial function . The development of similar models for CHCHD2P9 would depend on identifying orthologs in model organisms or creating transgenic animals expressing human CHCHD2P9. These models could be particularly valuable for investigating potential roles in disease processes, especially if CHCHD2P9 is found to have similar associations with neurodegenerative diseases as seen with CHCHD2 and CHCHD10 .

What is the potential role of CHCHD2P9 in disease processes?

While specific disease associations for CHCHD2P9 have not been extensively documented, the parent protein CHCHD2 has significant associations with several diseases, suggesting potential areas for investigation with CHCHD2P9. CHCHD2 mutations have been linked to Parkinson's Disease 22 (Autosomal Dominant) and Hereditary Essential Tremor 3 . These neurodegenerative disease associations highlight the importance of CHCHD2 in maintaining neuronal health, potentially through its roles in mitochondrial function and protection against oxidative stress.

Additionally, CHCHD2 overexpression has been implicated in multiple cancer types, including hepatocellular carcinoma, breast cancer, non-small cell lung cancer, and renal cell carcinoma . In these contexts, CHCHD2 appears to promote cancer cell survival, proliferation, and metastasis, potentially through its anti-apoptotic functions and effects on cellular metabolism. Elevated CHCHD2 levels correlate with poor prognosis in several cancers, suggesting it may serve as a prognostic biomarker . Whether CHCHD2P9 exhibits similar disease associations or potentially modulates CHCHD2-related disease processes represents an important area for future investigation.

How might CHCHD2P9 relate to mitochondrial dysfunction in neurodegenerative diseases?

The established role of CHCHD2 in neurodegenerative diseases suggests potential mechanisms by which CHCHD2P9 might also influence mitochondrial health and neurodegeneration. CHCHD2 mutations have been linked to Parkinson's disease, with evidence suggesting these mutations impair mitochondrial function through effects on crista structure and cytochrome c stability . Loss of CHCHD2 leads to mitochondrial dysfunction, increased reactive oxygen species (ROS) production, and enhanced susceptibility to apoptosis – all processes implicated in neurodegeneration.

If CHCHD2P9 produces a functional protein with structural similarity to CHCHD2, it might influence mitochondrial health through similar mechanisms or potentially modulate CHCHD2 function. Alternatively, as a pseudogene, CHCHD2P9 might regulate CHCHD2 expression through RNA-level interactions, as has been observed with other pseudogenes. Recent research has highlighted the importance of mitochondrial homeostasis in neurodegenerative diseases, with multiple mitochondrial proteins (including CHCHD2 and its "twin" protein CHCHD10) implicated in conditions like Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia . Understanding whether and how CHCHD2P9 fits into these pathways could provide valuable insights into disease mechanisms and potential therapeutic approaches.

What are the current challenges in differentiating between CHCHD2P9 and CHCHD2 in experimental settings?

One of the primary challenges in studying CHCHD2P9 is distinguishing it from CHCHD2 and other related proteins. This challenge exists at multiple levels, including genetic analysis, protein detection, and functional characterization. At the genetic level, the high sequence similarity between CHCHD2P9 and CHCHD2 can complicate PCR-based detection and quantification, requiring careful primer design to ensure specificity. RNA sequencing analyses must also employ specialized computational approaches to accurately distinguish reads mapping to highly similar sequences.

At the protein level, the development of specific antibodies that can distinguish between CHCHD2P9 and CHCHD2 represents a significant challenge but would be valuable for immunoblotting, immunoprecipitation, and immunohistochemistry studies. Without such specific tools, researchers must rely on epitope-tagged recombinant proteins, which may not fully recapitulate endogenous expression patterns and levels. Functional studies face similar challenges, as phenotypes observed upon manipulation of CHCHD2P9 could potentially result from indirect effects on CHCHD2 or other related proteins rather than direct effects of CHCHD2P9 itself. Addressing these challenges requires a combination of careful experimental design, validation using multiple complementary approaches, and potentially the development of new specific research tools.

How might CHCHD2P9 interact with the mitochondrial stress response pathways?

Based on what is known about CHCHD2, several potential mechanisms can be hypothesized for how CHCHD2P9 might influence mitochondrial stress responses. CHCHD2 has been shown to play important roles in regulating mitochondrial respiration, with effects on cytochrome c stability and oxidative phosphorylation . Additionally, CHCHD2 appears to function as a negative regulator of mitochondria-mediated apoptosis through its interaction with BCL-XL and inhibition of Bax oligomerization . These functions place CHCHD2 at a critical intersection of mitochondrial bioenergetics and cell death regulation.

If CHCHD2P9 produces a functional protein with similar domains, it might participate in similar processes or potentially modulate CHCHD2 function through competitive interactions. Research has also demonstrated connections between CHCHD2 and endoplasmic reticulum stress, with evidence that CHCHD2 may be part of a feedback mechanism that stimulates oxidative phosphorylation and promotes cell survival under stress conditions . This suggests potential roles in the communication between different cellular stress response pathways. Investigations into whether CHCHD2P9 participates in these stress response networks would provide valuable insights into cellular adaptation to mitochondrial dysfunction and might identify new therapeutic targets for diseases characterized by mitochondrial stress.

What novel research directions should be considered for CHCHD2P9 investigation?

Future research on CHCHD2P9 would benefit from several innovative approaches to address fundamental questions about its expression, structure, and function. One priority would be comprehensive transcriptomic analyses across diverse tissues and disease states to determine whether CHCHD2P9 is actively transcribed and under what conditions its expression might be regulated. This could include analysis of single-cell RNA sequencing data to identify cell type-specific expression patterns that might be masked in bulk tissue analyses.

Structural biology approaches comparing CHCHD2P9 to CHCHD2 and other CHCH domain proteins would provide valuable insights into potential functional similarities or differences. If CHCHD2P9 is confirmed to produce a protein, detailed characterization of its interactome using approaches like proximity labeling (BioID or APEX) coupled with mass spectrometry could reveal unique interaction partners distinct from those of CHCHD2. Additionally, the potential regulatory relationship between CHCHD2P9 and CHCHD2 should be investigated, as pseudogenes can sometimes regulate their parent genes through various RNA-level mechanisms. Finally, given the associations of CHCHD2 with both neurodegenerative diseases and cancer, examining CHCHD2P9 expression and potential functions in these disease contexts could uncover novel pathogenic mechanisms or therapeutic targets.

Table 1: Comparative Features of Key CHCH Domain Proteins

The CHCH domain protein family includes several important mitochondrial proteins, with CHCHD2 and CHCHD10 being the best characterized members. These "twin" proteins share structural similarities with their characteristic CHCH domains but have distinct and sometimes overlapping functions . CHCHD2 functions as a transcription factor that can translocate from mitochondria to the nucleus under stress conditions, where it regulates the expression of genes involved in oxidative phosphorylation . Additionally, CHCHD2 plays important roles in inhibiting mitochondria-mediated apoptosis and maintaining mitochondrial crista structure .

CHCHD10, the closest paralog to CHCHD2, is similarly localized to the mitochondrial intermembrane space and contributes to maintaining crista integrity and regulating oxidative phosphorylation . Mutations in both CHCHD2 and CHCHD10 have been linked to neurodegenerative diseases, though with somewhat different disease spectra – CHCHD2 mutations are primarily associated with Parkinson's disease, while CHCHD10 mutations have been linked to a broader range of conditions including amyotrophic lateral sclerosis and frontotemporal dementia . The specific characteristics of CHCHD2P9 relative to these better-studied family members remain to be fully elucidated, representing an important area for future research.

What research findings support potential functional roles for CHCHD2P9?

Particularly intriguing is the evidence that CHCHD2 serves as a modulator for scavenging reactive oxygen species (ROS) and can undergo ROS-dependent translocation from mitochondria to the nucleus . This dynamic subcellular localization enables CHCHD2 to coordinate nuclear and mitochondrial responses to stress. Additionally, the finding that CHCHD2 is localized to both the endoplasmic reticulum and mitochondria suggests potential roles in ER-mitochondria communication . Whether CHCHD2P9 retains any of these functions or perhaps modulates CHCHD2 activity through competitive interactions remains an open question. The increasing recognition that some pseudogenes produce functional proteins or regulatory RNAs further supports the possibility that CHCHD2P9 might have biological significance despite its classification as a pseudogene.

What are the most pressing unanswered questions about CHCHD2P9?

The current state of knowledge about CHCHD2P9 leaves several fundamental questions unanswered, creating opportunities for significant research contributions. First and foremost is the basic question of whether CHCHD2P9 is expressed at appreciable levels in human tissues, either as a protein or as a non-coding RNA with regulatory functions. Comprehensive expression profiling across tissues, cell types, and disease states would provide crucial baseline information for subsequent functional studies. If expressed, determining the subcellular localization of CHCHD2P9 would be essential for understanding its potential functions.

Another critical area for investigation is the structural characterization of CHCHD2P9, particularly in comparison to CHCHD2. Determining whether CHCHD2P9 maintains the characteristic CHCH domain structure with properly formed disulfide bonds would provide insights into its potential functional capabilities. Additionally, identifying potential interaction partners of CHCHD2P9 would be valuable for understanding its biological roles. Of particular interest would be whether CHCHD2P9 interacts with CHCHD2 itself, potentially modulating its functions. Finally, given the associations of CHCHD2 with neurodegenerative diseases and cancer, investigating whether CHCHD2P9 has similar disease associations could reveal new pathogenic mechanisms and therapeutic targets.

How might emerging technologies advance CHCHD2P9 research?

Emerging technologies offer promising approaches to address the current knowledge gaps regarding CHCHD2P9. Advanced RNA sequencing technologies, including long-read sequencing platforms like PacBio and Oxford Nanopore, could improve the detection and quantification of CHCHD2P9 transcripts, particularly in distinguishing them from the highly similar CHCHD2 transcripts. Single-cell RNA sequencing would enable the identification of cell type-specific expression patterns that might be masked in bulk tissue analyses. Spatial transcriptomics approaches could further reveal the tissue distribution of CHCHD2P9 expression with unprecedented resolution.

For protein-level analyses, advanced proteomics approaches like data-independent acquisition mass spectrometry might enable the detection of low-abundance CHCHD2P9 protein, if expressed. Proximity labeling methods such as BioID or APEX, combined with mass spectrometry, could identify the protein interaction network of CHCHD2P9 in living cells. CRISPR-based technologies offer powerful tools for both loss-of-function and gain-of-function studies to investigate the cellular consequences of CHCHD2P9 manipulation. Finally, integrative multi-omics approaches that combine transcriptomic, proteomic, and metabolomic data could provide a systems-level understanding of how CHCHD2P9 might influence cellular physiology, particularly in the context of mitochondrial function and stress responses.