CDC26 Human

What is CDC26 and what are its primary functions in human cells?

CDC26, also known as ANAPC12 or APC12, is a protein-coding gene (Entrez Gene ID: 246184) that encodes a component of the anaphase-promoting complex/cyclosome (APC/C). This complex functions as a cell cycle-regulated ubiquitin-protein ligase responsible for the controlled proteolysis of various cell cycle regulatory proteins .

The primary function of CDC26 is to contribute to the structural integrity of the APC/C complex. As one of the smaller subunits, CDC26 helps stabilize interactions between larger subunits, particularly by inserting into the TPR (tetratricopeptide repeat) grooves of APC6 . This structural support is essential for maintaining proper APC/C function throughout the cell cycle.

CDC26 is highly similar to Saccharomyces cerevisiae Cdc26, indicating evolutionary conservation of this important regulatory component . The APC/C complex as a whole is responsible for targeting specific substrates for ubiquitin-mediated degradation by the 26S proteasome, making it crucial for cell cycle progression, particularly during the metaphase-to-anaphase transition and mitotic exit .

How is CDC26 structurally organized within the anaphase-promoting complex?

Specifically, CDC26 is anchored at its N-terminus by inserting into the TPR grooves of APC6 . This interaction serves to stabilize the TPR groove through the formation of protein-protein interactions. The extended structure of CDC26 allows it to bridge and reinforce connections between multiple subunits within the complex.

What experimental techniques are available for detecting CDC26 expression?

Several experimental techniques have been validated for detecting CDC26 expression in research settings:

• Quantitative PCR (qPCR): This technique allows precise quantification of CDC26 mRNA levels. Validated primers for human CDC26 include:

  Gene	Primer sequence 5′-3′	Amplicon
  CDC26-Human	F: 5′-AGGAGGCAGTGATGGAGAAGGA-3′	111 bp
  	R: 5′-CGATTATTGGGCTTGGGTTGGG-3′
  18S-Human (reference)	F: 5′-ACAGGATTGACAGATTGA-3′	226 bp
  	R: 5′-TATCGGAATTAACCAGACA-3′

  These primers have been successfully used to compare CDC26 expression between young and aged oocytes .

• Immunocytochemistry: This method enables visualization of CDC26 protein localization within cells. It has been effectively employed to analyze CDC26 protein levels and distribution in oocytes .

• Single-cell RNA sequencing: This powerful technique allows comprehensive analysis of gene expression at the single-cell level. It has been used to identify differential gene expression, including CDC26, between young and old women's oocytes .

• Western blotting: Though not explicitly mentioned in the search results, this standard protein detection method would typically be used to quantify CDC26 protein levels in cell or tissue extracts.

When studying CDC26 in model organisms such as mice, species-specific primers should be used:

These methodological approaches provide researchers with validated tools for studying CDC26 expression across different experimental contexts .

How does CDC26 function differ between model organisms and humans?

Comparative studies reveal both conservation and divergence in CDC26 function across species:

In C. elegans:

• The CDC26 homolog (identified as B0511.9/cdc-26) functions as a component of the anaphase-promoting complex .

• Inhibition of cdc-26 activity leads to embryonic polarity defects after weak knockdown and meiotic metaphase I arrest following strong knockdown, similar to defects observed after inhibition of other APC/C subunits .

• C. elegans CDC-26 binds to APC/C component EMB-27/Cdc16, consistent with its role in stabilizing APC/C subunit interactions .

In S. cerevisiae (budding yeast):

• While human and S. cerevisiae APC/C architectures share common features, there are significant differences in the structures of smaller subunits like CDC26 .

• A notable regulatory difference exists: in S. cerevisiae, the catalytic module APC2:APC11 adopts an active conformation even without coactivator presence, whereas human APC/C requires coactivator binding to induce the active conformation .

• Mechanisms of APC/C CDC20 activation by phosphorylation appear to differ between yeast and humans .

In humans:

• CDC26 is critical for oocyte quality, with decreased expression in aged oocytes correlating with increased aneuploidy .

• Human CDC26 forms part of a 12-subunit APC/C complex (compared to 13 in yeast) .

• Human CDC26 can be experimentally overexpressed to partially rescue age-related defects in oocytes .

These comparative analyses highlight that while the core function of CDC26 as part of the APC/C is evolutionarily conserved, species-specific adaptations exist in terms of structure, regulation, and specialized functions. These differences must be considered when translating findings from model organisms to human applications .

What is the relationship between CDC26 expression and oocyte aging?

Research has established a significant relationship between CDC26 expression and oocyte aging with important implications for reproductive biology:

Single-cell RNA-seq analysis comparing oocytes from young and older women revealed that CDC26 mRNA and protein levels are severely decreased in aged oocytes . This reduction correlates with functional consequences in reproductive capacity.

The most significant finding is that aged oocytes lacking CDC26 demonstrate increased susceptibility to aneuploidy (abnormal chromosome numbers) . Since aneuploidy is a primary cause of infertility, miscarriage, and developmental disorders, this establishes CDC26 as a key molecular factor in age-related fertility decline.

Importantly, experimental evidence shows that these defects in aged oocytes can be partially rescued through overexpression of the CDC26 protein . This was demonstrated using recombinant lentiviral vectors encoding CDC26 transfected into germinal vesicle oocytes of older women, resulting in improved oocyte development .

These findings suggest a causal relationship where decreased CDC26 expression contributes directly to the compromised oocyte quality observed with advanced maternal age. The mechanisms likely involve disruption of APC/C complex function, which is essential for proper chromosome segregation during meiosis .

This research establishes CDC26 as a potential therapeutic target for addressing age-related decline in oocyte quality and female fertility, though additional studies are needed to fully elucidate the downstream molecular pathways through which CDC26 regulates oocyte maturation .

What are the current hypotheses regarding CDC26's role in preventing aneuploidy during oocyte maturation?

Several mechanistic hypotheses have been proposed to explain CDC26's role in preventing aneuploidy during oocyte maturation:

• APC/C Structural Integrity Hypothesis: CDC26, as a component of the anaphase-promoting complex/cyclosome (APC/C), is crucial for maintaining the structural integrity of this complex . Reduced CDC26 expression in aged oocytes could lead to APC/C destabilization, impairing proper regulation of cell cycle proteins required for accurate chromosome segregation.

• Regulated Proteolysis Hypothesis: The APC/C functions as a cell cycle-regulated ubiquitin-protein ligase responsible for targeting specific proteins for degradation . CDC26 deficiency may disrupt this regulated proteolysis, leading to inappropriate accumulation of proteins that should be degraded during specific phases of meiosis, contributing to chromosomal missegregation.

• Spindle Assembly Regulation Hypothesis: Research has demonstrated that aged oocytes lacking CDC26 are more prone to aneuploidy . This suggests CDC26 may play a role in regulating spindle assembly or function during meiosis, with its decreased expression contributing to spindle defects that lead to chromosome segregation errors.

• Coactivator Interaction Hypothesis: CDC26 may facilitate proper interactions between the APC/C and its coactivators (like CDC20 or CDH1) . Disruption of these interactions in CDC26-deficient oocytes could impair the timing or substrate specificity of APC/C activity during meiotic divisions.

How can researchers experimentally manipulate CDC26 expression to study its effects on oocyte maturation?

Researchers have successfully employed several strategies to manipulate CDC26 expression for studying its role in oocyte maturation:

• Overexpression approaches:

  • Recombinant lentiviral vectors: Transfection of lentiviral vectors encoding CDC26 into germinal vesicle oocytes has been effectively used to increase CDC26 expression . This approach successfully demonstrated partial rescue of age-related defects in human oocytes.

  • mRNA microinjection: Though not explicitly mentioned in the search results, this is a standard technique for oocyte manipulation that could be adapted for CDC26 overexpression studies.

• Knockdown/knockout approaches:

  • RNA interference (RNAi): This technique has been successfully used to inhibit cdc-26 activity in C. elegans, resulting in specific phenotypes including embryonic polarity defects and meiotic metaphase I arrest . Different RNAi intensities (weak vs. strong) produced distinct phenotypes, allowing for nuanced functional analysis.

  • Feeding RNAi protocols: For C. elegans studies, established protocols include placing L4-stage worms on feeding plates for specific durations (20-40 hours at 20°C) to achieve different levels of gene knockdown .

• Experimental design considerations:

  • Timing of manipulation: For studying oocyte maturation effects, manipulation at the germinal vesicle stage has proven effective .

  • Controls: Proper experimental controls are essential, as demonstrated in C. elegans studies comparing cdc-26 RNAi effects with those of other APC/C components .

  • Phenotypic analysis: Multiple readouts should be assessed, including oocyte maturation rates, chromosome segregation, and aneuploidy incidence.

• Model system selection:

  • Human oocytes: Discarded human oocytes from fertility clinics provide the most directly relevant model but have ethical and availability limitations .

  • Mouse oocytes: Provide a mammalian model with greater experimental flexibility while maintaining relevance to human biology .

  • C. elegans: Offers advantages for high-throughput genetic screening and detailed phenotypic analysis .

• Analysis methods:

  • Videorecording analysis: This has been effectively used in C. elegans to document real-time effects of CDC26 manipulation on cell division processes .

  • Immunostaining: Techniques for antibody staining of key markers (e.g., PAR proteins in C. elegans) provide valuable insights into molecular consequences of CDC26 manipulation .

These methodological approaches offer researchers a comprehensive toolkit for investigating CDC26 function in oocyte maturation across different model systems, with each approach having specific advantages depending on the research question .

What analytical methods are most effective for detecting CDC26 protein-protein interactions within the APC/C complex?

Several complementary analytical methods have proven effective for investigating CDC26 protein-protein interactions within the APC/C complex:

• Cryo-electron microscopy (Cryo-EM):

  • Provides high-resolution structural information about the entire APC/C complex

  • Has been successfully used to define structures of both S. cerevisiae and human APC/C at atomic resolution (4.0 Å)

  • Reveals detailed interaction interfaces between CDC26 and other APC/C subunits

  • Enables comparative structural analysis between species

• Two-hybrid screening:

  • Large-scale two-hybrid studies in C. elegans have demonstrated that CDC-26 binds to APC/C component EMB-27/Cdc16

  • Can identify binary interactions between CDC26 and other proteins

  • Useful for mapping interaction networks within complex protein assemblies

• Structural prediction approaches:

  • AlphaFold2 has been successfully used to predict structures of APC/C components like APC9

  • Can guide experimental structural studies by providing initial models

  • Particularly valuable for predicting interactions involving proteins with limited experimental structural data

• Complementation assays:

  • Yeast complementation studies using expression vectors containing CDC26 homologs can test functional conservation

  • For example, C. elegans B0511.9a (CDC26 homolog) has been expressed in yeast using recombinant plasmids to assess functional complementation

  • Requires vectors with appropriate promoters (e.g., PGK promoter) and marker genes (e.g., URA3)

• Combined methodological approaches:

  • Integration of structural analysis with genetic and biochemical data provides the most comprehensive understanding

  • Sequential application of computational prediction followed by experimental validation is particularly effective

  • Cross-species comparisons can highlight conserved interaction interfaces with functional significance

Each method has specific strengths and limitations, and a comprehensive understanding of CDC26 interactions within the APC/C complex typically requires integration of multiple approaches. The selection of methods should be guided by the specific research question, available resources, and the nature of the interactions being studied .

What are the best experimental models for studying CDC26 function in human reproductive aging?

Several experimental models offer valuable insights into CDC26 function in reproductive aging, each with distinct advantages:

• Human oocyte models:

  • Discarded human oocytes: Provide the most directly relevant model for studying human CDC26 function in reproductive aging

  • Can be collected for single-cell RNA-seq, q-PCR, and immunocytochemical analyses to identify differential gene expression patterns

  • Limited availability requires careful experimental design to maximize data yield

  • In vitro maturation (IVM) of human oocytes:

    • Allows controlled experimental conditions while maintaining clinical relevance

    • Can be combined with CDC26 genetic manipulation through lentiviral transfection

    • Enables assessment of CDC26's effects on oocyte development and chromosomal stability

• Mouse models:

  • Mouse oocyte studies:

    • Provide a mammalian model with greater experimental flexibility

    • Allow for genetic manipulation and detailed developmental analysis

    • CDC26 primers for mouse studies have been validated:

      Gene	Primer sequence 5′-3′	Amplicon
      CDC26-Mouse	F: 5′-AGGAACCAGCGATGGAGAAGGAG-3′	118 bp
      	R: 5′-AGATGTGCGGTTGTTGCTCTTGAG-3′

• C. elegans models:

  • Powerful system for genetic analysis of cell division processes

  • RNA interference coupled with videorecording enables detailed phenotypic analysis

  • The CDC26 homolog (B0511.9/cdc-26) has been well-characterized

  • Allows high-throughput screening approaches not feasible in mammalian models

  • Established protocols for RNAi feeding (20-40 hours at 20°C) and phenotypic analysis

• Comparative experimental approach:

  • Integrating findings across multiple model systems provides the most comprehensive understanding

  • Initial mechanistic studies in C. elegans can identify key pathways for targeted investigation in mammalian models

  • Validation of key findings in human samples provides clinical relevance

A multi-model approach has proven most effective, beginning with mechanistic studies in genetically tractable organisms like C. elegans, continuing with functional validation in mouse models, and culminating with confirmation in human samples. This strategy balances experimental flexibility with clinical relevance and has successfully demonstrated CDC26's critical role in oocyte aging .

What potential therapeutic strategies could target CDC26 to address age-related fertility decline?

Based on current research findings, several promising therapeutic strategies targeting CDC26 could potentially address age-related fertility decline:

• Gene therapy approaches:

  • Lentiviral CDC26 delivery: Research has already demonstrated that transfection of recombinant lentiviral vectors encoding CDC26 into germinal vesicle oocytes can partially rescue age-related defects . This proof-of-principle finding suggests that gene therapy approaches could be developed to increase CDC26 expression in oocytes during IVF procedures.

  • Optimization opportunities: Future research could focus on improving vector design, enhancing transfection efficiency, and optimizing expression levels for maximum efficacy while maintaining safety.

• Oocyte screening and selection strategies:

  • CDC26 expression biomarkers: Developing methods to assess CDC26 expression levels in oocytes could enable selection of eggs with higher developmental potential during IVF procedures.

  • Integration with existing technologies: CDC26 screening could potentially complement current preimplantation genetic testing approaches to improve embryo selection.

• Development of modified culture conditions:

  • Culture media optimization: Identification of factors that help maintain CDC26 expression during in vitro oocyte maturation could lead to improved culture protocols.

  • Temporal regulation: Since CDC26 functions within the anaphase-promoting complex/cyclosome (APC/C), timing interventions to coincide with critical points in meiotic progression could maximize efficacy.

• Target upstream regulators:

  • Research to identify factors that regulate CDC26 expression could reveal additional therapeutic targets.

  • Pharmaceutical interventions targeting these regulators might provide alternatives to direct gene therapy approaches.

• Translational research considerations:

  • Safety evaluation: Any therapeutic intervention would require rigorous safety assessment, particularly given the critical nature of chromosome segregation in development.

  • Efficacy metrics: Clear endpoints for measuring intervention success need to be established, potentially including aneuploidy rates, embryo development parameters, and pregnancy outcomes.

  • Individualized approaches: Given the heterogeneity in reproductive aging, personalized approaches based on individual CDC26 expression profiles might maximize therapeutic benefit.

While these strategies show promise based on current research, significant additional work is needed to translate these findings into clinical applications. Future studies should focus on optimizing delivery methods, establishing safety profiles, and conducting controlled trials to determine efficacy in human fertility treatment settings .

What are the most promising future research directions for CDC26 in reproductive biology?

Several promising research directions emerge from current understanding of CDC26's role in reproductive biology:

• Mechanistic Understanding of CDC26 Function:

  • Elucidating the complete molecular pathway through which CDC26 deficiency leads to aneuploidy in aged oocytes

  • Identifying direct molecular targets of CDC26 within the context of oocyte maturation

  • Investigating whether additional signaling pathways interact with CDC26 to regulate oocyte quality

• Therapeutic Development for Fertility Preservation:

  • Optimizing lentiviral vector systems for CDC26 delivery to maximize efficacy and safety

  • Investigating whether CDC26-based interventions could improve IVF outcomes in older women

  • Exploring the potential of CDC26 as a biomarker for oocyte quality assessment

• Comparative and Evolutionary Biology:

  • Further comparative studies between human and model organism APC/C structures and functions

  • Investigation of species-specific adaptations in CDC26 function that may reveal additional therapeutic targets

  • Exploring whether CDC26 regulation differs in species with unusual reproductive longevity

• Integrative Approaches:

  • Combining structural biology insights from cryo-EM studies with functional genomics approaches

  • Developing comprehensive models of how CDC26 functions within the broader network of reproductive aging

  • Multi-omics studies comparing young and aged oocytes to identify co-regulated networks involving CDC26

• Translational Research:

  • Development of clinically applicable methods to assess CDC26 function as potential diagnostic tools

  • Evaluating whether CDC26 expression correlates with clinical IVF outcomes across different age groups

  • Investigating whether other APC/C components might serve as complementary therapeutic targets

These research directions hold promise for advancing both fundamental understanding of reproductive biology and development of interventions to address age-related fertility decline, a significant issue in reproductive medicine with broad societal implications .

How might understanding CDC26 function lead to innovative approaches for assessing and improving oocyte quality?

Understanding CDC26 function could lead to several innovative approaches for assessing and improving oocyte quality: