Recombinant Mouse Protein aurora borealis (Bora)

What is Aurora Borealis (Bora) protein and what is its primary function?

Aurora Borealis (Bora) is a conserved mitotic protein that functions as a critical co-activator of Aurora A kinase and plays a key role in activating Polo-like kinase 1 (PLK1). First identified in Drosophila melanogaster, Bora controls the proper timing of mitosis onset by enabling Aurora A to activate PLK1 . In mammals, this protein is essential for cell cycle progression, particularly during the G2/M transition. Bora's activity is tightly regulated during the cell cycle, with its function being particularly crucial during mitotic entry. Mechanistically, Bora binds to Aurora A and enhances its kinase activity, which subsequently leads to proper centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis .

How conserved is Bora across different species?

Bora exhibits remarkable evolutionary conservation from C. elegans to humans, indicating its fundamental role in eukaryotic cell division . The functional domains of Bora, particularly those involved in Aurora A binding and activation, show high sequence similarity across species. Both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro, demonstrating functional conservation . The conserved region in the N-terminal portion is critical for Aurora-A binding, while the non-conserved C-terminus appears dispensable for this interaction. This evolutionary conservation suggests that Bora-mediated regulation of Aurora A represents a fundamental mechanism in mitotic control that has been maintained throughout eukaryotic evolution.

What is the subcellular localization pattern of Bora during the cell cycle?

Bora exhibits a dynamic subcellular localization pattern that changes throughout the cell cycle:

• In interphase cells: Bora is predominantly a nuclear protein

• Upon entry into mitosis: Bora is excluded from the nucleus and translocates to the cytoplasm

• This nuclear export is Cdc2-dependent

This localization pattern is functionally significant as it provides a spatial regulatory mechanism for Aurora A activation. During interphase, nuclear retention of Bora helps keep Aurora A inactive. When Cdc2 (CDK1) becomes activated at the G2/M transition, Bora is released into the cytoplasm where it can bind and activate Aurora A . This mechanism elegantly explains how sequential activation of Cdc2 leads to Aurora A activation, establishing a temporal coordination of mitotic events.

How does Bora contribute to PLK1 activation during mitotic entry?

Bora serves as a critical intermediary in the activation cascade of PLK1 during mitotic entry through several coordinated mechanisms:

• CDK1-mediated phosphorylation: Under recovery conditions from G2/M checkpoint, CDK1 (Cdc2) phosphorylates Bora on its N-terminal domain, which is essential for PLK1 re-activation and subsequent mitotic commitment .

• Aurora A facilitation: Bora binds to Aurora A and enhances its kinase activity, which then phosphorylates PLK1 at threonine 210 in its activation loop.

• Conformational changes: The binding of Bora to the kinase domain of Aurora A may induce conformational changes that facilitate PLK1 access to the Aurora A active site.

• Cytoplasmic translocation: As Bora translocates from the nucleus to the cytoplasm during prophase (in a Cdc2-dependent manner), it enables spatial coordination of PLK1 activation at specific subcellular locations .

This multilayered activation mechanism ensures proper timing of PLK1 activity, which is crucial for centrosome maturation, bipolar spindle formation, and chromosome segregation during mitosis.

What happens to cell cycle progression when Bora is depleted or overexpressed?

Effects of Bora depletion:

• Cell cycle arrest or delay at G2/M transition

• Reduced PLK1 activation and decreased phosphorylation of its downstream targets

• Apoptosis-consistent pattern of condensed and/or fragmented chromatin

• Reduction in the surrogate marker of PLK1 activity (pTCTP-Ser46)

• Defects in asymmetric cell division similar to those observed in Aurora-A mutants

• Impaired centrosome maturation and spindle assembly

Effects of Bora overexpression:

• Enhanced proliferation rate in both normal and cancer cells

• Increased capacity to form colonies in soft agar, indicating malignant transformation potential

• Loss of contact inhibition, with cells forming multiple layers after reaching confluence

• Can rescue defects caused by mutations in Aurora-A

• Accelerated migration capacity (approximately two-fold increase)

These opposing phenotypes highlight Bora's central role in regulating mitotic progression and cellular transformation, making it a potential target for anticancer therapeutic strategies.

What are the optimal expression systems for producing recombinant mouse Bora protein?

For the production of functional recombinant mouse Bora protein, several expression systems have been validated with varying advantages:

For most research applications requiring high functional activity, the baculovirus-insect cell system offers the best balance between yield and proper protein processing. For in vitro binding assays where post-translational modifications are less critical, bacterial expression using MBP or GST fusion tags has been successfully employed to produce Bora that can bind and activate Aurora A .

What are the validated methods for assessing Bora-Aurora A interaction in vitro?

Several robust methodologies have been established to assess Bora-Aurora A interactions:

• Co-immunoprecipitation (Co-IP):

  • Transfect cells with tagged Aurora A and Bora (e.g., Bora-GFP)

  • Lyse cells and perform immunoprecipitation using anti-tag antibodies

  • Detect interaction by Western blotting

• GST/MBP pull-down assays:

  • Express GST-Bora or MBP-Bora fusion proteins in bacteria

  • Incubate with in vitro translated Aurora A

  • Analyze binding by SDS-PAGE and autoradiography or Western blotting

• In vitro kinase assays:

  • Express and purify Aurora A and Bora from bacteria or insect cells

  • Incubate Bora with Aurora A in the presence of ATP

  • Measure phosphorylation by autoradiography or phospho-specific antibodies

  • This approach can assess both binding and functional activation simultaneously

• Surface Plasmon Resonance (SPR):

  • Immobilize purified Bora or Aurora A on sensor chips

  • Measure real-time binding kinetics with the partner protein

  • Determine association/dissociation constants

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag Bora and Aurora A with compatible fluorophores

  • Measure energy transfer upon interaction in vitro or in live cells

The optimal method should be selected based on the specific research question, with a combination of approaches providing the most robust validation of interaction.

How is Bora implicated in ovarian cancer progression and what are the experimental models to study this?

Bora has been identified as having a significant oncogenic role in ovarian cancer (OC) through multiple lines of evidence:

Ovarian Cancer Implications:

Experimental Models for Studying Bora in Ovarian Cancer:

These models collectively provide complementary approaches to investigate Bora's role in ovarian cancer, with the combination of in vitro, in vivo, and clinical specimen analysis offering the most comprehensive understanding of its oncogenic functions.

What are the potential therapeutic strategies targeting Bora or its pathway in cancer?

Several promising therapeutic strategies targeting Bora or its associated pathways have emerged from recent research:

• Direct Bora Inhibition:

  • While specific small molecule inhibitors of Bora are still under development, RNA interference approaches have demonstrated that Bora depletion can impair cancer cell viability in vitro, in vivo, and in patient-derived samples

• Targeting Downstream Effectors:

  • Combinatory treatments using FDA-approved inhibitors against oncogenic downstream effectors of Bora have shown synergistic effects in ovarian cancer models

  • Specifically, combined inhibition of BCL2 and CDK6 (identified as Bora downstream effectors) has demonstrated remarkable therapeutic potential

• Aurora A Inhibitors:

  • Since Bora activates Aurora A, Aurora A inhibitors (several in clinical trials) may indirectly block Bora-dependent pathways

  • This approach may be particularly effective in cancers with Bora overexpression

• PLK1 Inhibitors:

  • As Bora is critical for PLK1 activation, PLK1 inhibitors represent another strategy to block the Bora-dependent oncogenic pathway

  • Examples include volasertib and BI2536

• Cell Cycle Checkpoint Modulators:

  • Targeting the G2/M checkpoint in combination with Bora pathway inhibition may create synthetic lethality in cancer cells

• Combination Approaches:

  • Based on transcriptome analysis of Bora-depleted cells, rational combinations targeting survival, dissemination, and inflammation-related pathways offer promising avenues

These therapeutic strategies highlight the potential of targeting the Bora pathway as a novel approach for cancer treatment, particularly in tumors showing high Bora expression, such as advanced ovarian cancers.

How do post-translational modifications regulate Bora activity and stability?

Bora's activity and stability are tightly regulated through multiple post-translational modifications (PTMs) that ensure precise temporal control of its function during cell cycle progression:

• CDK1-mediated phosphorylation:

  • CDK1 phosphorylates Bora on its N-terminal domain

  • This phosphorylation is essential for PLK1 reactivation and mitotic commitment

  • Serves as a key regulatory event linking CDK1 activation to PLK1 activation

• ATR-mediated phosphorylation:

  • Under DNA damage conditions, ATR (ataxia telangiectasia and Rad3 related kinase) phosphorylates Bora

  • This phosphorylation marks Bora for degradation

  • Sustains G2/M checkpoint blockade by preventing PLK1 activation

• Ubiquitin-dependent degradation:

  • Bora levels are regulated through the ubiquitin-proteasome system

  • SCF-β-TrCP E3 ubiquitin ligase complex has been implicated in Bora degradation

  • Degradation timing is critical for proper mitotic progression

• Aurora A-mediated phosphorylation:

  • Creates a feedback loop where Aurora A may phosphorylate Bora

  • This may affect Bora's activity or stability

These modification events form an intricate regulatory network that coordinates Bora function with cell cycle progression and DNA damage responses. Understanding these PTMs provides potential intervention points for therapeutic development targeting Bora-dependent pathways in cancer.

What is the structural basis for Bora-mediated activation of Aurora A?

The structural basis for Bora-mediated activation of Aurora A involves specific domain interactions and conformational changes:

• Critical Binding Regions:

  • The N-terminal region of Bora is essential for Aurora A binding

  • Deletion studies have shown that removing either the conserved region (BoraΔ2) or a region N-terminal to the conserved part (BoraΔ1) abrogates the interaction with Aurora A

  • The C-terminus of Bora (beyond amino acid 404 in Drosophila Bora) is dispensable for Aurora A binding and activation

• Activation Mechanism:

  • Bora binding likely induces conformational changes in Aurora A's activation loop

  • This conformational change enhances Aurora A's catalytic activity toward substrates like PLK1

  • The interaction may also protect the activating phosphorylation of Aurora A (Thr288 in humans) from dephosphorylation

• Species Conservation:

  • The binding interface is highly conserved, as evidenced by cross-species interaction capabilities

  • Human Aurora A can bind to Drosophila MBP-Bora in vitro, demonstrating structural conservation

  • This conservation suggests fundamental structural requirements for the interaction

• Comparison with Other Activators:

  • Unlike TPX2 (another Aurora A activator), which prevents PP1-dependent dephosphorylation, Bora appears to have a more direct activation mechanism

  • Bora may represent a more universal activation pathway for Aurora A that is conserved across species

While detailed structural data from crystallography or cryo-EM studies of the Bora-Aurora A complex are still pending, these biochemical insights provide a framework for understanding the molecular basis of this critical regulatory interaction in mitotic progression.

What are the optimal conditions for in vitro kinase assays using recombinant Bora and Aurora A?

For robust and reproducible in vitro kinase assays with recombinant Bora and Aurora A, the following optimized conditions are recommended:

Reagent Preparation:

• Recombinant Aurora A: Best expressed in insect cells with a removable His-tag

• Recombinant Bora: Can be expressed as MBP-fusion protein from bacteria or insect cells

• Both proteins should be purified to >90% homogeneity by affinity chromatography followed by size exclusion

Reaction Components:

Controls to Include:

• Aurora A alone without Bora (baseline activity)

• Heat-inactivated Aurora A (negative control)

• Known Aurora A activator such as TPX2 (positive control)

• Catalytically inactive Aurora A mutant (e.g., K162R)

Detection Methods:

• Radioactive assay: Use [γ-³²P]ATP and detect incorporation by autoradiography or scintillation counting

• Western blot: Use phospho-specific antibodies against Aurora A (pT288) or substrate

• ELISA-based: For higher throughput quantification

• ADP-Glo™: Luminescence-based detection of ADP production

These optimized conditions should provide a reliable system to assess Bora's ability to activate Aurora A and can be adapted for inhibitor screening or mechanistic studies.

How can researchers effectively model Bora-dependent pathways in 3D cell culture systems?

3D cell culture systems offer significant advantages over traditional 2D cultures for studying Bora-dependent pathways in a more physiologically relevant context. Here are methodological approaches for effectively modeling these pathways:

Recommended 3D Culture Systems:

Analytical Methods for 3D Systems:

• Live Imaging Approaches:

  • Confocal microscopy with Bora-fluorescent protein fusions

  • Time-lapse imaging to track mitotic progression

  • Light-sheet microscopy for deeper tissue penetration

• Molecular Analysis:

  • RNA isolation from 3D cultures using specialized extraction protocols

  • Protein extraction with enhanced lysis buffers containing stronger detergents

  • Single-cell sequencing for heterogeneity assessment

• Functional Assays:

  • Growth curve analysis using 3D image quantification

  • Invasion/migration assays in 3D matrices

  • Cell cycle analysis by EdU incorporation and flow cytometry

  • Apoptosis detection using cleaved caspase-3 staining

• Genetic Manipulation Strategies:

  • Inducible shRNA or CRISPR systems for temporal control

  • Viral transduction protocols optimized for 3D structures

  • Region-specific genetic manipulation for spatial control

These methodologies enable researchers to investigate Bora's functions in contexts that better recapitulate the in vivo environment, particularly important for understanding its roles in processes like asymmetric cell division, which are highly dependent on three-dimensional cellular organization.

What emerging technologies might advance our understanding of Bora's function in development and disease?

Several cutting-edge technologies are poised to significantly advance our understanding of Bora's functions:

• CRISPR-based Technologies:

  • Base editing and prime editing: For introducing precise mutations in Bora to study structure-function relationships

  • CRISPRi/CRISPRa systems: For temporal control of Bora expression in specific tissues

  • CRISPR screens: To identify synthetic lethal interactions with Bora in cancer cells

• Advanced Imaging Technologies:

  • Super-resolution microscopy: To visualize Bora-Aurora A interactions at nanometer resolution

  • Lattice light-sheet microscopy: For long-term imaging of Bora dynamics during development

  • FRET/FLIM techniques: To measure real-time protein interactions in living cells and tissues

• Single-cell Multi-omics:

  • Single-cell RNA-seq with CITE-seq: To correlate Bora expression with protein markers across cell populations

  • Single-cell proteomics: To analyze Bora pathway activity at individual cell resolution

  • Spatial transcriptomics: To map Bora expression patterns in intact tissues

• Structural Biology Approaches:

  • Cryo-EM: To determine the structure of Bora-Aurora A complexes

  • Hydrogen-deuterium exchange mass spectrometry: To map dynamic conformational changes

  • AlphaFold2/RoseTTAFold: For computational prediction of Bora structure and interactions

• Organoid and In Vivo Technologies:

  • Patient-derived organoids: To study Bora function in personalized cancer models

  • Tissue-specific conditional knockouts: For developmental studies in mice

  • In vivo CRISPR screens: To identify context-dependent functions

• Optical Control Technologies:

  • Optogenetics: For spatial and temporal control of Bora activity

  • Photo-caged compounds: To activate or inhibit Bora function with light

  • Opto-FGFR: For light-controlled activation of signaling pathways upstream of Bora

These emerging technologies, especially when used in combination, have the potential to provide unprecedented insights into Bora's functions in development, cell cycle regulation, and cancer biology.

How might the understanding of Bora contribute to precision medicine approaches in cancer therapy?

The growing understanding of Bora's functions presents several promising avenues for precision medicine approaches in cancer therapy:

• Biomarker Development:

  • Bora expression levels correlate with aggressive disease in ovarian cancer

  • The human Bora homolog is located on chromosome 13 in a region associated with breast cancer susceptibility

  • These observations suggest Bora could serve as a prognostic biomarker

  • Quantitative assessment of Bora protein or mRNA in biopsies could help stratify patients for treatment selection

• Patient Stratification Strategies:

  • Patients with high Bora expression may benefit from targeted therapies against the Bora-Aurora A-PLK1 axis

  • Transcriptome analysis could identify tumors with activated Bora-dependent pathways

  • Combining Bora expression with other cell cycle markers could create treatment-predictive signatures

• Novel Therapeutic Targets:

  • Downstream effectors of Bora (BCL2, CDK6) represent actionable targets with existing FDA-approved inhibitors

  • The synergistic effect observed when targeting these effectors offers promising combination therapy approaches

  • Rational drug combinations based on Bora pathway activation could maximize efficacy while minimizing toxicity

• Combination Therapy Design:

  • Transcriptome analysis of Bora-depleted cells revealed modulated genes involved in survival, dissemination, and inflammation-related pathways

  • This knowledge enables rational design of combination therapies targeting multiple aspects of Bora's oncogenic functions

  • Example combinations include Aurora A inhibitors with anti-apoptotic protein inhibitors or immune modulators

• Drug Resistance Mechanisms:

  • Understanding how Bora-dependent pathways contribute to therapy resistance

  • Developing strategies to overcome resistance by targeting Bora or its downstream effectors

  • Monitoring Bora pathway activation as a mechanism of acquired resistance

• Innovative Therapeutic Approaches:

  • Protein-protein interaction disruptors targeting the Bora-Aurora A interface

  • Degraders (PROTACs) targeting Bora for selective degradation

  • Cell cycle phase-specific delivery systems that exploit Bora's cell cycle-dependent regulation

As research advances, these precision medicine approaches could significantly improve outcomes for patients with cancers driven by dysregulation of the Bora-Aurora A-PLK1 axis, particularly in ovarian and potentially breast cancers where Bora's oncogenic role has been established.