CDC34 Human

What is human CDC34 and what is its primary function in cellular processes?

Human CDC34 (also known as UBE2R1) is an E2 ubiquitin-conjugating enzyme that plays a crucial role in the ubiquitin-proteasome system by catalyzing the formation of polyubiquitin chains on target proteins. CDC34 functions primarily in conjunction with the SCF E3 ubiquitin ligase complex to mediate the ubiquitination of proteins involved in cell cycle regulation . The enzyme accepts activated ubiquitin from an E1 enzyme and subsequently transfers it to target proteins recognized by the SCF complex, marking them for degradation by the 26S proteasome . Human CDC34 is highly conserved evolutionarily, as evidenced by its ability to complement growth defects in temperature-sensitive yeast CDC34 mutant strains (cdc34-2), demonstrating the functional conservation of this enzyme across species .

What are the key structural domains of human CDC34 and how do they contribute to its function?

Human CDC34 contains several functionally distinct domains:

• N-terminal catalytic core domain: Contains the active site cysteine residue that forms the thioester bond with ubiquitin .

• C-terminal tail: Critical for CDC34 function and contains multiple important regions:

  • UBS1 (Ubiquitin Binding Site 1): Spans residues 206-215 and contains aromatic residues (Phe206, Tyr207, Tyr210, and Tyr211) that interact with ubiquitin .

  • UBS2 (Ubiquitin Binding Site 2): Spans residues 216-225 and also binds ubiquitin .

  • Acidic tail region: Contains a stretch of acidic residues (Asp/Glu) beyond position 226 .

The contribution of these domains to CDC34's function is evidenced by complementation studies showing that human CDC34(1-215) containing UBS1 can partially restore growth in yeast cdc34-2 mutants, while CDC34(1-200) lacking UBS1 cannot support growth. The full-length CDC34 with both UBS1 and UBS2 provides optimal function , as summarized in the table below:

How does human CDC34 interact with ubiquitin molecules during the ubiquitination process?

Human CDC34 interacts with ubiquitin through two distinct C-terminal motifs: UBS1 (residues 206-215) and UBS2 (residues 216-225) . These interactions were identified using NMR chemical shift perturbation assays with 15N-labeled CDC34 C-terminal fragment and ubiquitin. The binding affinity (Kd) for both UBS1 and UBS2 is approximately 1.6-1.7 mM .

The interaction between CDC34 and ubiquitin exhibits several important characteristics:

• Binding specificity: UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys48 and the C-terminal tail, which are key sites for conjugation .

• Key residues: The aromatic residues in UBS1 (Phe206, Tyr207, Tyr210, and Tyr211) are positioned near ubiquitin's C-terminal residue Val70. Mutations of these aromatic residues to glycine or changing ubiquitin Val70 to alanine decreases binding affinity .

• Condition sensitivity: The interactions are sensitive to both pH and salt concentration, with optimal binding occurring at acidic pH. Addition of NaCl at 500 mM abolishes the interactions .

• Functional significance: Reconstituted IκBα ubiquitination analysis revealed that the UBS1 aromatic residues contribute to conjugation activity, with Tyr210 playing a particularly important role. CDC34 Tyr210 is required for the transfer of donor ubiquitin to receptor lysine residues in a manner dependent on the neddylated RING subcomplex of SCF .

What mechanisms regulate the activation of human CDC34 in the context of SCF-mediated ubiquitination?

Human CDC34 activation involves several sophisticated regulatory mechanisms:

• Proximity-induced activation: Research has shown that the mere juxtaposition of CDC34 molecules can dramatically enhance its catalytic activity. This was demonstrated through experiments with GST-fused CDC34, which forms dimers and shows constitutively high activity in supporting K48-linked polyubiquitin chain assembly, independent of SCF .

• Chemical-induced dimerization: When CDC34 was fused to FK506-binding protein (FKBP) and treated with the chemical inducer AP20187, the induced dimeric form exhibited substantially higher activity than the monomeric form in catalyzing ubiquitin-ubiquitin ligation .

• SCF-mediated activation: The SCF core ubiquitin ligase module, composed of the ROC1 RING finger protein and the CUL1 C-terminus with a Nedd8 moiety conjugated at K720, dramatically enhances CDC34's activity in ubiquitin-ubiquitin ligation reactions .

• Transition from inactive monomer to active dimer: Evidence suggests that SCF-mediated polyubiquitination may require the conversion of CDC34 from an inactive monomeric form to a highly active dimeric form, potentially explaining how SCF enhances CDC34's catalytic capability .

These findings collectively suggest that spatial organization of CDC34 enzymes is a key regulatory mechanism that controls their activity in polyubiquitin chain formation, potentially offering new insights for therapeutic intervention.

How does the neddylation of Cullin proteins affect CDC34 function in the SCF complex?

Neddylation of Cullin proteins plays a critical role in regulating CDC34 function within the SCF complex:

• Enhanced catalytic activity: The presence of a Nedd8 moiety covalently conjugated to Cullin-1 (CUL1) at K720 substantially enhances CDC34's ubiquitin-ubiquitin ligation activity . This neddylated CUL1 C-terminus, in complex with the ROC1 RING finger protein, forms a core ubiquitin ligase module that dramatically activates CDC34 .

• Substrate ubiquitination dependence: Particularly noteworthy is that CDC34 Tyr210 is required for the transfer of donor ubiquitin to receptor lysine residues in a manner that depends specifically on the neddylated RING subcomplex of SCF . This suggests that structural changes induced by neddylation create an optimal environment for CDC34-mediated ubiquitin transfer.

• Conformational effects: Neddylation likely induces conformational changes in the SCF complex that optimize the positioning of CDC34 relative to both the donor ubiquitin and the substrate, enhancing the efficiency of ubiquitin transfer.

This neddylation-dependent regulation represents a sophisticated control mechanism that ensures CDC34 activity is precisely regulated in the context of SCF-mediated ubiquitination, providing an additional layer of specificity to the ubiquitin-proteasome system.

What are the molecular mechanisms underlying CDC34's specificity for K48-linked polyubiquitin chain formation?

CDC34 specifically catalyzes the formation of K48-linked polyubiquitin chains, which typically target proteins for proteasomal degradation. This specificity is determined by several molecular mechanisms:

• Ubiquitin binding orientation: The UBS1 and UBS2 domains of CDC34 bind to ubiquitin in the proximity of ubiquitin Lys48, positioning this residue optimally for conjugation to the C-terminus of another ubiquitin molecule .

• Structural positioning: When bound to ubiquitin, the CDC34 UBS1 aromatic residues (Phe206, Tyr207, Tyr210, and Tyr211) are positioned near ubiquitin's C-terminal residue Val70, facilitating the transfer of ubiquitin to a receptor lysine .

• Catalytic specificity: CDC34 Tyr210 plays a particularly important role in the transfer of donor ubiquitin specifically to Lys48 of a receptor ubiquitin, demonstrating how specific residues in CDC34 contribute to chain linkage specificity .

• SCF-dependent orientation: The interaction with the SCF complex, particularly the neddylated RING subcomplex, further enhances CDC34's specificity for K48-linkage formation by properly orienting the donor ubiquitin relative to the acceptor lysine .

These mechanisms collectively ensure that CDC34 efficiently catalyzes the formation of K48-linked polyubiquitin chains, providing specificity to the ubiquitin-proteasome system's targeting of proteins for degradation.

What are the most effective experimental approaches for studying human CDC34 structure and interactions?

Researchers have employed several effective experimental approaches to study human CDC34:

• NMR Chemical Shift Perturbation (CSP) Assays:

  • Successfully identified direct interactions between CDC34 C-terminus and ubiquitin

  • Mapped binding interfaces to specific residues (UBS1 and UBS2)

  • Determined binding constants (Kd values of 1.6-1.7 mM for UBS1 and UBS2)

  • Assessed the effects of pH and salt concentration on interactions

• 3D Structural Analysis:

  • Obtained 3D structural snapshots of CDC34 in action

  • Revealed key features important for cell growth regulation and activity

  • Identified unique structural features relevant for drug design

• Yeast Complementation Assays:

  • Evaluated the function of human CDC34 domains in vivo

  • Tested various truncated constructs (CDC34(1-200), CDC34(1-215), full-length) for their ability to complement temperature-sensitive cdc34-2 yeast strain

  • Demonstrated the functional importance of specific domains

• Reconstituted Ubiquitination Assays:

  • Analyzed the role of specific residues in catalytic function

  • Identified Tyr210 as particularly important for ubiquitin transfer

  • Demonstrated dependence on neddylated RING subcomplex

• Dimerization Studies:

  • Used GST fusion to create constitutively dimeric CDC34

  • Employed chemical induction of dimerization with FKBP-CDC34 fusion and AP20187

  • Demonstrated the importance of dimerization for CDC34 activity

These methodologies provide complementary information about CDC34 structure, function, and regulation, offering researchers multiple approaches to investigate different aspects of this important enzyme.

How can researchers effectively express and purify human CDC34 for biochemical and structural studies?

Based on successful experimental approaches described in the research literature, here is a methodological guide for expressing and purifying human CDC34:

• Expression Systems:

  • Bacterial expression: E. coli BL21(DE3) has been successfully used to express recombinant human CDC34 with various tags (His, GST, FLAG)

  • Fusion tags: N-terminal fusion tags such as His6, GST, or FKBP can be used depending on the experimental requirements

  • Expression constructs: Both full-length and truncated versions (e.g., CDC34C spanning residues 170-236) can be expressed for different analyses

• Purification Strategy:

  • Affinity chromatography: Use tag-specific methods (Ni-NTA for His-tagged proteins, glutathione sepharose for GST-fusion proteins)

  • Size exclusion chromatography: For obtaining highly purified protein and separating different oligomeric states

  • Ion exchange chromatography: Particularly useful given CDC34's charged regions

• Protein Labeling for Structural Studies:

  • For NMR studies, 15N-labeling of CDC34 can be achieved by growing bacteria in minimal media with 15NH4Cl as the sole nitrogen source

  • This approach was successfully used to produce 15N-labeled CDC34C for interaction studies with ubiquitin

• Buffer Optimization:

  • CDC34-ubiquitin interactions are sensitive to pH and salt concentration

  • Optimal interactions occur at acidic pH

  • High salt (500 mM NaCl) abolishes CDC34-ubiquitin interactions

  • Buffer composition should be optimized based on the specific experimental requirements

• Quality Control:

  • Assess protein folding using circular dichroism

  • Verify activity using in vitro ubiquitination assays

  • Confirm oligomeric state by size exclusion chromatography or analytical ultracentrifugation

These methodological approaches provide a foundation for researchers to efficiently produce human CDC34 protein suitable for a wide range of biochemical and structural studies.

What assays are most informative for measuring CDC34 activity in vitro and in cellular systems?

Researchers have employed several complementary assays to measure CDC34 activity across different experimental systems:

In Vitro Assays:

• Ubiquitin-Ubiquitin Ligation Assays:

  • Measures CDC34's ability to catalyze the formation of polyubiquitin chains

  • Demonstrates the enhancement of activity by SCF core ubiquitin ligase module

  • Can be used to evaluate the effect of different CDC34 mutations on catalytic activity

• Reconstituted IκBα Ubiquitination Analysis:

  • Evaluates CDC34-catalyzed ubiquitination of physiological substrates

  • Allows assessment of the role of specific residues (e.g., Phe206/Tyr207, Tyr210/Tyr211) in conjugation

  • Demonstrates dependence on neddylated RING subcomplex

• NMR-Based Binding Assays:

  • Uses chemical shift perturbation to detect interactions between CDC34 and ubiquitin

  • Enables determination of binding constants and mapping of interaction interfaces

  • Sensitive to conditions such as pH and salt concentration

Cellular Assays:

• Yeast Complementation Assays:

  • Tests the ability of human CDC34 constructs to rescue growth defects in temperature-sensitive cdc34-2 yeast strains

  • Allows evaluation of the functional significance of different CDC34 domains in vivo

  • Demonstrated that CDC34(1-215) containing UBS1 partially complements, while CDC34(1-200) without UBS1 fails to complement

• Dominant Negative Studies:

  • Uses CDC34 mutants (e.g., C93S and L97S) that interfere with endogenous CDC34 function

  • Helps evaluate the consequences of CDC34 inhibition in cellular systems

• Antisense Expression:

  • Depletes endogenous CDC34 using antisense constructs

  • Allows assessment of CDC34's role in specific cellular processes

• Expression Analysis During Development:

  • Tracks CDC34 expression patterns during developmental processes

  • Revealed developmentally regulated expression patterns of CDC34, Rad6B, and Cullin proteins in murine testicular development

  • Showed elevation in meiotic and postmeiotic haploid germ cells

These diverse assays provide comprehensive insights into CDC34 activity across different experimental contexts, from purified protein systems to complex cellular environments, allowing researchers to investigate various aspects of CDC34 function.

How is CDC34 dysregulated in cancer and what are the implications for cancer biology?

CDC34 dysregulation in cancer has significant implications for understanding cancer biology and developing therapeutic strategies:

• Cell Cycle Regulation: CDC34 plays a critical role in regulating cell cycle progression through the ubiquitination and subsequent degradation of key cell cycle proteins. Dysregulation of CDC34 can lead to abnormal accumulation of these proteins, promoting uncontrolled cell proliferation characteristic of cancer .

• Target Protein Stabilization: When CDC34 function is compromised, its target proteins, including those involved in transcriptional regulation and cell cycle control, may be stabilized inappropriately. For example, CDC34 targets repressors of cAMP-induced transcription, and its dysfunction could alter transcriptional regulation in cancer cells .

• Developmental Connection: Studies have shown that CDC34, along with Rad6B and Cullin proteins, is expressed in a developmentally regulated manner, with elevated levels in meiotic and postmeiotic haploid germ cells. This suggests that CDC34 dysregulation could contribute to developmental abnormalities associated with certain cancers .

• SCF Complex Interaction: CDC34 functions in conjunction with the SCF E3 ubiquitin ligase complex. Alterations in this interaction, which might result from mutations in either CDC34 or components of the SCF complex, could disrupt normal protein degradation pathways relevant to cancer progression .

The specific mechanisms of CDC34 dysregulation in cancer provide insights into fundamental cancer biology and highlight potential targets for therapeutic intervention, making CDC34 an important subject for cancer research.

What structural features of human CDC34 could be exploited for the rational design of specific inhibitors?

Human CDC34 possesses several unique structural features that make it an attractive target for rational drug design:

• C-terminal Ubiquitin Binding Sites:

  • UBS1 (residues 206-215) and UBS2 (residues 216-225) bind to ubiquitin near its Lys48 and C-terminal tail

  • These sites are critical for CDC34 function and could be targeted by small molecules to disrupt CDC34-ubiquitin interactions

  • The aromatic residues in UBS1 (Phe206, Tyr207, Tyr210, and Tyr211) are particularly important for function and could serve as specific binding pockets for inhibitors

• Dimerization Interface:

  • CDC34 activation involves a transition from inactive monomer to active dimer

  • Compounds that prevent dimerization could potentially inhibit CDC34 activity

  • This approach might offer greater specificity than targeting the catalytic site, which is more conserved among E2 enzymes

• SCF Interaction Surfaces:

  • CDC34 interacts with the SCF complex, particularly the neddylated RING subcomplex

  • Disrupting these protein-protein interactions could specifically inhibit CDC34-mediated ubiquitination

  • The dependence of CDC34 Tyr210 on the neddylated RING subcomplex for ubiquitin transfer suggests that this interaction could be a target for inhibition

• 3D Structural Features:

  • Recently obtained 3D structural snapshots have revealed key features of CDC34 important for its regulation of cell growth and activity

  • These structural insights provide opportunities for structure-based drug design approaches

• Condition-Sensitive Interactions:

  • CDC34-ubiquitin interactions are sensitive to pH and salt concentration

  • This sensitivity could potentially be exploited to design inhibitors that function under specific cellular conditions

These structural features provide multiple potential avenues for the development of specific CDC34 inhibitors, which could have therapeutic applications in cancer treatment where CDC34 activity is dysregulated.

What approaches have been used to validate CDC34 as a therapeutic target in cancer research?

Several approaches have been employed to validate CDC34 as a potential therapeutic target in cancer research:

• Structural and Functional Characterization:

  • 3D structural snapshots of CDC34 have revealed key features important for its regulation of cell growth and activity

  • These structural insights have identified unique features that could be targeted for cancer therapeutics

  • Understanding of the precise mechanisms by which CDC34 functions provides a rational basis for therapeutic intervention

• Dominant Negative and Antisense Studies:

  • Experiments using dominant negative mutants (C93S and L97S) of human CDC34 have helped evaluate the consequences of CDC34 inhibition

  • Antisense constructs targeting CDC34 have been used to assess its role in cellular processes relevant to cancer

  • These approaches demonstrate the effects of CDC34 inhibition on cancer-related cellular processes

• Interaction with Cancer-Relevant Substrates:

  • CDC34 has been shown to target repressors of cAMP-induced transcription, including regulatory factors relevant to cancer

  • Its role in the ubiquitination of IκBα and p27Kip1, both important in cancer biology, further validates its relevance as a therapeutic target

• Developmentally Regulated Expression:

  • Studies showing that CDC34 is expressed in a developmentally regulated manner, with elevated levels in specific cell types and stages, suggest its importance in cellular differentiation processes that may be dysregulated in cancer

• Cancer Context Validation:

  • Collaborative research between basic scientists and cancer biologists/clinicians has explored CDC34's role specifically in cancer contexts

  • Extension of molecular findings to biological contexts in human cancer cells validates CDC34 as a therapeutic target

These multi-faceted approaches collectively establish CDC34 as a promising therapeutic target in cancer research, providing a strong foundation for the development of specific inhibitors that could have clinical applications.

What are the current limitations in our understanding of human CDC34 biology?

Despite significant advances in CDC34 research, several important limitations remain in our understanding:

• Complete Structural Understanding:

  • While recent studies have provided 3D structural snapshots of CDC34 , a complete understanding of its structure, especially in different functional states (e.g., monomeric vs. dimeric, free vs. SCF-bound), remains elusive

  • The dynamic structural changes that occur during the catalytic cycle are not fully characterized

• Substrate Specificity Mechanisms:

  • The complete repertoire of CDC34 substrates in human cells remains undefined

  • The precise mechanisms that determine substrate specificity, beyond the role of the SCF complex, are incompletely understood

  • How CDC34 distinguishes between different SCF complexes with various F-box proteins needs further investigation

• Tissue-Specific Functions:

  • While CDC34 expression has been studied in specific contexts like testicular development , its tissue-specific functions and regulation across different human tissues and developmental stages remain to be fully elucidated

• Regulatory Networks:

  • The integration of CDC34 function within broader cellular regulatory networks, including its interplay with other ubiquitin-conjugating enzymes and signaling pathways, needs further clarification

  • How CDC34 activity is modulated in response to various cellular stresses or stimuli is incompletely understood

• Post-translational Modifications:

  • The potential role of post-translational modifications in regulating CDC34 activity has not been comprehensively investigated

  • Whether CDC34 itself is regulated by ubiquitination or other modifications remains unclear

Addressing these limitations will require continued research using emerging technologies and interdisciplinary approaches to provide a more comprehensive understanding of CDC34 biology.

What emerging technologies or methodologies might advance our understanding of CDC34 function?

Several emerging technologies and methodologies hold promise for advancing our understanding of CDC34 function:

• Cryo-Electron Microscopy (Cryo-EM):

  • Could provide high-resolution structures of CDC34 in complex with SCF and substrates

  • May capture different conformational states during the ubiquitination cycle

  • Could visualize the structural basis of CDC34 dimerization and activation

• Single-Molecule Techniques:

  • Single-molecule FRET could track conformational changes during CDC34 catalytic cycles

  • Optical tweezers or atomic force microscopy could measure forces involved in CDC34-mediated ubiquitin transfer

  • These approaches would provide insights into the dynamic aspects of CDC34 function not captured by static structural studies

• Proximity Labeling Proteomics:

  • BioID or APEX2-based approaches could identify proteins in the vicinity of CDC34 in living cells

  • Would help map the complete CDC34 interactome under different cellular conditions

  • Could identify novel substrates and regulatory partners

• CRISPR-Based Genetic Screens:

  • Genome-wide CRISPR screens could identify genes that synthetically interact with CDC34

  • CRISPR-based base editing could introduce specific mutations to study structure-function relationships

  • Would provide insights into the broader cellular networks involving CDC34

• Quantitative Ubiquitinomics:

  • Mass spectrometry-based methods to quantitatively assess changes in the ubiquitinome upon CDC34 manipulation

  • Could identify the complete range of CDC34 substrates and ubiquitination sites

  • Would provide insights into the global impact of CDC34 on cellular protein homeostasis

• Integrative Structural Biology:

  • Combining multiple structural techniques (X-ray crystallography, NMR, Cryo-EM) with computational modeling

  • Could provide comprehensive structural models of CDC34 in different functional states

  • Would integrate dynamic and static structural information

• Organoid and In Vivo Models:

  • Human organoid models to study CDC34 function in a physiologically relevant context

  • Conditional knockout mouse models to assess tissue-specific functions

  • Would bridge the gap between molecular mechanisms and physiological relevance

These emerging approaches, especially when used in combination, have the potential to significantly advance our understanding of CDC34 function at molecular, cellular, and organismal levels.

How might research on CDC34 impact our broader understanding of the ubiquitin-proteasome system and cell cycle regulation?

Research on CDC34 has the potential to significantly impact our broader understanding of the ubiquitin-proteasome system (UPS) and cell cycle regulation in several important ways:

• Mechanistic Insights into E2-E3 Cooperation:

  • Studies showing that CDC34 is activated through dimerization and interaction with the SCF complex provide a paradigm for understanding how E2-E3 cooperation enhances ubiquitination efficiency

  • This could reveal general principles applicable to other E2-E3 pairs in the UPS

• Understanding Chain Specificity:

  • CDC34's specificity for K48-linked polyubiquitin chain formation, mediated by its UBS domains , offers insights into how linkage specificity is achieved in the UPS

  • Could help explain how different ubiquitin chain topologies lead to distinct cellular outcomes

• Regulation of Protein-Protein Interactions:

  • The identification of non-covalent ubiquitin binding sites in CDC34 highlights the importance of protein-protein interactions beyond the catalytic site

  • May reveal how secondary interactions enhance specificity and efficiency in ubiquitination reactions throughout the UPS

• Cell Cycle Checkpoint Integration:

  • CDC34's role in targeting cell cycle regulators for degradation connects the UPS directly to cell cycle checkpoint mechanisms

  • Research on CDC34 substrates could reveal how ubiquitin-mediated protein degradation is integrated with cell cycle progression

  • May identify novel regulatory mechanisms at cell cycle checkpoints

• Evolutionary Conservation of UPS Components:

  • The high conservation of CDC34 function across species, as demonstrated by complementation studies , provides insights into the evolutionary importance of specific UPS components

  • Helps identify core, conserved mechanisms in the UPS that are fundamental to eukaryotic cell function

• Therapeutic Targeting Strategies:

  • Approaches to target CDC34 for cancer therapeutics could establish principles for targeting other UPS components

  • May lead to novel strategies for modulating protein degradation pathways in disease contexts

• Developmental Regulation:

  • The developmental regulation of CDC34 expression, particularly in processes like spermatogenesis , highlights how the UPS is dynamically regulated during development

  • Could reveal how cell cycle regulation is adapted to meet the needs of specialized developmental processes

By advancing our understanding in these areas, CDC34 research serves as a model system that illuminates broader principles governing the UPS and cell cycle regulation, with implications for both basic science and therapeutic applications.

What are the most significant recent advances in CDC34 research?

Recent CDC34 research has yielded several significant advances that have transformed our understanding of this important enzyme:

• Structural Insights:

  • Obtaining 3D structural snapshots of CDC34 has revealed key features important for its regulation of cell growth and activity, providing foundations for rational drug design

  • Identification of specific ubiquitin binding sites (UBS1 and UBS2) in the C-terminal region of CDC34 has elucidated how CDC34 interacts with ubiquitin during the catalytic cycle

• Activation Mechanisms:

  • Discovery that CDC34 is activated through dimerization has revolutionized our understanding of its catalytic mechanism

  • The finding that juxtaposition of CDC34 molecules dramatically enhances activity suggests that SCF-mediated polyubiquitination may require conversion of CDC34 from an inactive monomer to an active dimer

• Functional Domain Mapping:

  • Precise mapping of functional domains in CDC34, particularly the C-terminal region containing UBS1 (residues 206-215) and UBS2 (residues 216-225), has clarified structure-function relationships

  • Identification of key aromatic residues (Phe206, Tyr207, Tyr210, and Tyr211) that are critical for CDC34 function

• SCF Interaction Mechanisms:

  • Elucidation of how the neddylated RING subcomplex of SCF enhances CDC34 activity, particularly the dependence of CDC34 Tyr210 on this complex for ubiquitin transfer

  • These findings have clarified the molecular basis of E2-E3 cooperation in the ubiquitination process

• Therapeutic Potential:

  • Recognition of CDC34 as a promising target for cancer therapeutics, based on its role in cell cycle regulation and the identification of unique structural features that could be exploited for drug design

These advances collectively represent significant progress in understanding the molecular mechanisms of CDC34 function and its potential as a therapeutic target, laying the groundwork for future research and applications.

How can researchers best contribute to addressing the remaining questions about CDC34?

Researchers can contribute to addressing remaining questions about CDC34 through several strategic approaches:

• Interdisciplinary Collaboration:

  • Form collaborative teams that combine expertise in structural biology, biochemistry, cell biology, and clinical research

  • The most impactful CDC34 research has emerged from collaborations between basic scientists and cancer biologists/clinicians

  • This approach ensures that molecular insights are extended to relevant biological and clinical contexts

• Technological Integration:

  • Employ complementary methodologies to address different aspects of CDC34 biology

  • Combine structural approaches (X-ray crystallography, NMR, Cryo-EM) with functional assays (in vitro ubiquitination, yeast complementation) and cellular studies

  • Integrate emerging technologies such as proximity labeling proteomics and quantitative ubiquitinomics

• Systematic Mutational Analysis:

  • Conduct comprehensive structure-function studies through systematic mutation of key residues

  • Focus particularly on the aromatic residues in UBS1 (Phe206, Tyr207, Tyr210, and Tyr211) that have been shown to be important for CDC34 function

  • Extend these studies to understand the functional significance of other domains and residues

• Physiological Context:

  • Study CDC34 function in physiologically relevant contexts, such as development and tissue-specific functions

  • Build on existing knowledge of CDC34's developmentally regulated expression in systems like testicular development

  • Develop and utilize appropriate in vivo models to validate findings from in vitro and cellular studies

• Therapeutic Development:

  • Leverage structural insights to design specific CDC34 inhibitors

  • Focus on unique features such as the UBS domains and dimerization interface

  • Test candidate inhibitors in appropriate cancer models to validate CDC34 as a therapeutic target

• Systems-Level Analysis:

  • Place CDC34 in the broader context of the ubiquitin-proteasome system and cell cycle regulation

  • Identify connections to other cellular pathways and processes

  • Use network analysis approaches to understand CDC34's role in cellular homeostasis