CCDC90B Human

What is CCDC90B and where is it located in the human genome?

CCDC90B (Coiled-coil domain containing 90B) is a protein encoded by the CCDC90B gene located on chromosome 11 in humans. It belongs to the CCDC90 protein family and has several aliases including MDS011, MDS025, and CUA003 . The gene is positioned in proximity to several other genes including PCF11 (a mammalian pre-mRNA cleavage complex 2 protein), ANKRD42 (an ankyrin repeat protein involved with calcium ion bonding), BC070093, and DLG2 (a member of the membrane-associated guanylate kinase family) . The genomic sequence is available in multiple reference assemblies including GRCh38.p14 Primary Assembly (NC_000011.10) and GRCh37.p13 Primary Assembly (NC_000011.9) .

For researchers initiating studies on CCDC90B, it is recommended to consult genomic databases such as UCSC Genome Browser for detailed gene structure analysis and to utilize NCBI's Gene database for comprehensive genetic information. Comparative genomic analyses across species may provide evolutionary insights into CCDC90B function.

What is the structural composition of the CCDC90B protein?

The CCDC90B protein is characterized by the presence of a domain of unknown function called DUF1640. This domain constitutes the majority of the protein with the exception of the first twenty-three amino acid residues (MNSRQAWRLFLSQGRGDRWVSRP), which function as a mitochondrial targeting signal and are cleaved during protein processing .

Structurally, CCDC90B contains seven predicted alpha helices, which is a characteristic feature of coiled-coil domain proteins . The mature protein (after cleavage of the mitochondrial targeting sequence) has the following physical properties:

For structural analysis, researchers should consider using techniques such as X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy to further elucidate the three-dimensional arrangement of the DUF1640 domain and its functional implications.

What are the predicted post-translational modifications of CCDC90B?

CCDC90B undergoes several post-translational modifications that may be critical to its function. The primary modification is the cleavage of the N-terminal mitochondrial targeting sequence (first 23 amino acids) . Additionally, the protein is predicted to contain at least three specific phosphorylation sites:

• Protein Kinase C (PKC) phosphorylation sites

• Casein Kinase II phosphorylation sites

• cAMP/cGMP-dependent phosphorylation sites

Other predicted post-translational modifications include:

For experimental validation of these predicted modifications, researchers should consider employing mass spectrometry-based proteomics, phospho-specific antibodies, and site-directed mutagenesis to understand the functional relevance of these modifications in different cellular contexts.

What methodological approaches are most effective for studying CCDC90B localization and function in mitochondria?

Given that CCDC90B is predicted to be a mitochondrial protein with a cleaved N-terminal targeting sequence, several complementary approaches can be employed to study its localization and function:

• Subcellular Fractionation and Western Blotting: Isolate mitochondrial fractions from cells expressing CCDC90B and confirm its presence using antibodies against the mature protein. Compare with markers for other mitochondrial compartments (outer membrane, intermembrane space, inner membrane, matrix) to determine precise localization.

• Fluorescence Microscopy: Generate fusion constructs of CCDC90B with fluorescent proteins (ensuring the tag doesn't interfere with the mitochondrial targeting sequence) and co-localize with mitochondrial markers such as MitoTracker dyes or antibodies against known mitochondrial proteins.

• Immunoelectron Microscopy: For high-resolution localization within mitochondrial subcompartments.

• Proximity Labeling Methods: Techniques such as BioID or APEX2 fusion proteins can identify proximal interacting partners in the mitochondrial environment.

• Loss-of-Function Studies: CRISPR/Cas9-mediated knockout or RNA interference to assess the impact of CCDC90B depletion on mitochondrial morphology, membrane potential, respiratory function, and metabolic parameters.

For recombinant protein work, researchers can utilize commercially available CCDC90B proteins such as the one described by ProSpec, which is produced in E. coli as a single, non-glycosylated polypeptide chain with a 23 amino acid His-tag at the N-terminus .

What experimental systems are optimal for investigating the DUF1640 domain of CCDC90B?

The Domain of Unknown Function 1640 (DUF1640) that characterizes CCDC90B presents a challenging research target. To elucidate its function:

• Comparative Genomics: Identify proteins with similar DUF1640 domains across species to infer evolutionary conservation and potential functional significance. The presence of CCDC90B orthologs in other species such as Xenopus tropicalis suggests evolutionary conservation .

• Domain Swapping Experiments: Replace the DUF1640 domain with analogous domains from related proteins to determine functional equivalence.

• Point Mutation Analysis: Create a library of mutations throughout the DUF1640 domain to identify critical residues for function.

• Structural Biology Approaches: X-ray crystallography or cryo-EM studies of the isolated domain to determine its three-dimensional structure.

• Interaction Mapping: Use techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions within DUF1640 that participate in protein-protein interactions.

The recombinant CCDC90B protein available from commercial sources can serve as a starting point for in vitro studies of DUF1640 domain properties, including stability, oligomerization state, and binding affinities .

What are the implications of CCDC90B phosphorylation for mitochondrial function?

CCDC90B is predicted to contain at least three types of phosphorylation sites (PKC, Casein Kinase II, and cAMP/cGMP-dependent), suggesting regulation by multiple signaling pathways . To investigate the functional consequences of CCDC90B phosphorylation:

• Phosphomimetic and Phospho-deficient Mutants: Generate versions of CCDC90B with serine/threonine residues mutated to aspartate/glutamate (phosphomimetic) or alanine (phospho-deficient) at predicted phosphorylation sites.

• Kinase Inhibitor Studies: Treat cells with specific inhibitors for PKC, Casein Kinase II, or PKA/PKG to observe effects on CCDC90B phosphorylation state and mitochondrial function.

• Phosphoproteomics: Use mass spectrometry-based approaches to identify specific phosphorylation sites on CCDC90B under different cellular conditions.

• Cell Signaling Context: Investigate how cellular stresses, metabolic states, or signaling events affect CCDC90B phosphorylation and subsequent mitochondrial outcomes.

When designing such experiments, consider using physiologically relevant cell types with high mitochondrial content (e.g., cardiomyocytes, neurons, hepatocytes) to capture the functional impact of CCDC90B phosphorylation on energy metabolism.

How should researchers approach the purification and handling of recombinant CCDC90B for in vitro studies?

When working with recombinant CCDC90B protein, researchers should consider the following methodological details:

• Expression Systems: The commercially available recombinant CCDC90B is produced in E. coli as a non-glycosylated polypeptide with a His-tag for purification . For studies requiring post-translational modifications, consider mammalian or insect cell expression systems.

• Purification Protocol: CCDC90B can be purified using proprietary chromatographic techniques, typically involving immobilized metal affinity chromatography (IMAC) due to the His-tag .

• Storage Conditions: Store purified CCDC90B at 4°C if using within 2-4 weeks, or at -20°C for longer periods. For extended storage, addition of a carrier protein (0.1% HSA or BSA) is recommended. Multiple freeze-thaw cycles should be avoided .

• Buffer Composition: The optimal buffer for CCDC90B stability contains 20mM Tris-HCl (pH 8.0), 0.15M NaCl, 10% glycerol, and 1mM DTT, with a typical protein concentration of 0.25mg/ml .

• Quality Control: Verify protein purity (>90%) using SDS-PAGE and confirm identity using mass spectrometry or western blotting .

For functional studies, researchers should consider the cleaved mature form of the protein (without the mitochondrial targeting sequence) to replicate the biologically active state within mitochondria.

What methodologies can elucidate the potential role of CCDC90B in human disease models?

While the search results don't explicitly mention CCDC90B's involvement in specific diseases, its mitochondrial localization suggests potential roles in mitochondrial disorders. Researchers could:

• Gene Expression Analysis: Compare CCDC90B expression levels across healthy tissues and disease states using RT-qPCR, RNA-seq, or mining public databases like GTEx and TCGA.

• Genetic Association Studies: Analyze whether CCDC90B variants are associated with mitochondrial disorders, metabolic diseases, or cancer predisposition. The NCBI database indicates there are variants documented in ClinVar .

• Disease Model Systems: Develop and characterize CCDC90B knockout or knockdown models in relevant cell lines or organisms to observe phenotypic consequences.

• Rescue Experiments: In disease models with CCDC90B dysfunction, attempt rescue with wild-type or modified CCDC90B to establish causality.

• Patient-Derived Samples: Analyze CCDC90B expression, localization, and post-translational modifications in relevant patient samples compared to controls.

For these studies, researchers should consider both molecular readouts (protein expression, mitochondrial function) and physiological parameters (cellular metabolism, viability, stress response) to comprehensively assess CCDC90B's contribution to disease phenotypes.

What techniques are recommended for studying CCDC90B interactions with neighboring genes on chromosome 11?

The CCDC90B gene is located on chromosome 11 in proximity to several other genes including PCF11, ANKRD42, BC070093, and DLG2 . To investigate potential functional relationships:

• Chromosome Conformation Capture (3C, 4C, Hi-C): These techniques can identify physical interactions between the CCDC90B gene locus and other genomic regions, potentially revealing co-regulated gene clusters.

• Transcriptomic Analysis: RNA-seq of cells after CCDC90B modulation can reveal whether neighboring genes show coordinated expression changes.

• Chromatin Immunoprecipitation (ChIP): Identify shared transcription factors or epigenetic marks across CCDC90B and neighboring genes.

• CRISPR Interference/Activation: Target the regulatory regions of CCDC90B to assess effects on neighboring gene expression.

• Synteny Analysis: Compare the genomic organization of CCDC90B and neighboring genes across species to identify evolutionarily conserved gene clusters.

Given that neighboring genes like PCF11 (involved in mRNA processing) and DLG2 (part of the MAGUK family) have distinct functions , any functional relationships might indicate novel biological pathways worthy of further investigation.

What are the optimal approaches for generating antibodies or probes specific to CCDC90B?

Researchers requiring specific detection tools for CCDC90B should consider:

• Epitope Selection: Choose unique regions of CCDC90B that differ from related proteins, particularly avoiding the conserved DUF1640 domain if specificity between family members is desired.

• Antibody Development Strategies:

  • Use synthetic peptides corresponding to unique CCDC90B regions

  • Use recombinant full-length protein (available commercially)

  • Consider developing antibodies that specifically recognize the phosphorylated forms of CCDC90B

• Validation Methods:

  • Western blotting against recombinant protein and endogenous CCDC90B

  • Immunoprecipitation followed by mass spectrometry

  • Immunocytochemistry with appropriate positive and negative controls

  • Testing in CCDC90B knockout/knockdown systems

• Non-antibody Probes:

  • CRISPR-based tagging of endogenous CCDC90B

  • Fluorescent protein fusion constructs

  • Proximity labeling constructs (BioID, APEX)

For researchers working with model organisms like Xenopus tropicalis, which has a CCDC90B ortholog , cross-reactivity testing is essential when using human CCDC90B-specific tools in comparative studies.

How should researchers design experiments to distinguish CCDC90B from related family members?

The CCDC90 family includes multiple members with similar structural features. To ensure specificity when studying CCDC90B:

• Sequence Alignment Analysis: Perform detailed sequence comparisons between CCDC90B and related proteins to identify unique regions for targeting.

• Specific Primer/Probe Design: For nucleic acid detection, design primers and probes that target unique regions of CCDC90B mRNA.

• Isoform-Specific Antibodies: Develop antibodies against regions that differ between CCDC90B and related proteins.

• Genetic Manipulation Specificity: When using RNAi or CRISPR techniques, thoroughly validate target specificity through rescue experiments and off-target effect assessment.

• Expression Pattern Analysis: Characterize the tissue-specific expression profiles of CCDC90B versus related family members to identify systems where one predominates.

When publishing research on CCDC90B, explicit description of the methods used to ensure specificity will strengthen the reliability and reproducibility of findings.

What are the current gaps in CCDC90B research that present opportunities for novel investigations?

Based on the available information, several knowledge gaps represent valuable research opportunities:

• Functional Characterization of DUF1640: The domain of unknown function that characterizes CCDC90B remains poorly understood. Structure-function studies could provide significant insights.

• Interactome Mapping: Comprehensive identification of CCDC90B-interacting proteins would illuminate its role in mitochondrial and cellular processes.

• Regulatory Mechanisms: The functional significance of the multiple predicted phosphorylation sites requires experimental validation.

• Physiological Role: The specific contribution of CCDC90B to mitochondrial function, particularly in different tissues and under various stress conditions, remains to be elucidated.

• Disease Associations: Potential links between CCDC90B variants or expression levels and human diseases, particularly mitochondrial disorders, represent an important area for investigation.

• Evolutionary Conservation: Comparative studies across species, including examination of the Xenopus tropicalis ortholog , could reveal evolutionarily conserved functions.