Recombinant Rat Spindle and centriole-associated protein 1 (Spice1), partial

What is SPICE1 and what are its primary cellular functions?

SPICE1 (Spindle and Centriole-Associated Protein 1, also known as SPICE or CCDC52) is a protein-coding gene involved in several critical cellular processes. Functionally, SPICE1 participates in metaphase chromosome alignment, mitotic spindle assembly, and regulation of centriole replication . This protein plays a pivotal role in ensuring proper cell division by maintaining genomic stability during mitosis.
The protein contains two significant coiled-coil domains (CC1 and CC2), with the CC1 domain being particularly important for its interactions with other proteins such as Fatty Acid Synthase (FASN) . When studying recombinant rat SPICE1, researchers should consider these structural domains as they are essential for the protein's functionality.

What is the genomic context of SPICE1?

SPICE1 is located on chromosome 3 at position 3q13.2. The specific genomic coordinates in the human reference genome (NC_000003.12) are 113,442,718 to 113,515,156 on the complement strand . The gene contains 18 exons, making it a moderately complex gene structure for expression studies .
When working with recombinant rat SPICE1, it's important to note that while the protein shares homology with human SPICE1, species-specific differences may exist in genomic organization that could affect experimental design and interpretation, particularly for cross-species studies.

Where is SPICE1 protein localized within cells?

SPICE1 demonstrates specific subcellular localization patterns that reflect its functional roles. The protein is predominantly found in three key cellular structures:

• Centrioles - where it participates in centriole duplication and elongation

• Centrosomes - where it contributes to centrosome function during cell division

• Mitotic spindle - where it facilitates proper chromosome alignment
  Immunofluorescence studies have been used to confirm these localization patterns. When designing experiments with recombinant rat SPICE1, researchers should consider co-localization studies with other centrosomal and spindle markers to validate proper protein targeting and function.

How is SPICE1 expression measured in experimental settings?

SPICE1 expression can be measured at both the mRNA and protein levels using several established techniques:

What experimental approaches can be used to study SPICE1 function in mitotic spindle assembly?

Investigating SPICE1's role in mitotic spindle assembly requires sophisticated methodological approaches:

• CRISPR-Cas9 gene editing: Generate SPICE1 knockout or domain-specific mutant cell lines to observe effects on spindle formation. This approach allows for precise genetic manipulation to study domain-specific functions.

• Live-cell imaging with fluorescently tagged SPICE1: Transfect cells with GFP-SPICE1 constructs to track protein dynamics during mitosis. Time-lapse microscopy can reveal the temporal association of SPICE1 with the mitotic spindle.

• Spindle assembly assays: Measure spindle formation efficiency in the presence or absence of recombinant SPICE1 protein. Quantify spindle length, morphology, and microtubule density using immunofluorescence and image analysis software.

• Phosphorylation studies: Since SPICE1 is a substrate for phosphorylation by aurora kinase , use phospho-specific antibodies or mass spectrometry to identify phosphorylation sites and their functional significance in spindle assembly.

• Co-immunoprecipitation: Identify novel SPICE1 interaction partners within the spindle apparatus. This technique has successfully identified the interaction between SPICE1 and CEP120, which facilitates procentriole elongation .
  These approaches should include appropriate controls and statistical analyses to ensure robust and reproducible results.

How does SPICE1 regulate centriole replication and what methods can investigate this mechanism?

SPICE1 participates in centriole duplication through several mechanisms that can be studied using these methodological approaches:

• Centrosome duplication assays: Treat cells with hydroxyurea to uncouple centrosome duplication from the cell cycle, then quantify centriole numbers using centrosome markers (e.g., γ-tubulin, centrin) in cells with modulated SPICE1 expression.

• Super-resolution microscopy: Employ techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to visualize centriole ultrastructure and SPICE1 localization at nanometer resolution.

• Proximity ligation assays (PLA): Detect protein-protein interactions between SPICE1 and other centriole proteins in situ, providing spatial context for these interactions within the centrosome.

• In vitro reconstitution: Purify recombinant SPICE1 and centriole components to reconstitute aspects of centriole assembly in cell-free systems, allowing for controlled manipulation of protein concentrations and conditions.
  Research has shown that SPICE1 interacts with CEP120 to facilitate procentriole elongation and recruitment of capping proteins essential for procentriole formation . Disrupting this interaction leads to abnormalities in centriole duplication, highlighting SPICE1's critical role in this process.

What is the relationship between SPICE1 and oncogenesis, particularly in osteosarcoma?

Recent research has identified SPICE1 as a potential oncogene in osteosarcoma (OS), with several experimental findings supporting this relationship:

• Expression analysis: SPICE1 is significantly overexpressed in OS tissues compared to normal tissues, with high expression correlating with poor patient prognosis .

• Functional studies: Both in vitro and in vivo experiments demonstrate that SPICE1 promotes OS cell proliferation and tumor growth:

  • SPICE1 knockdown significantly reduced cell proliferation in HOS and 143B osteosarcoma cell lines

  • SPICE1 overexpression enhanced proliferation as measured by CCK8, colony formation, and EdU assays

  • In nude mice models, SPICE1 knockdown inhibited tumor growth while SPICE1 overexpression accelerated tumor growth

• Molecular mechanism: SPICE1 enhances OS growth by stabilizing Fatty Acid Synthase (FASN) through inhibition of its ubiquitination. Specifically, SPICE1:

  • Interacts with FASN through its CC1 domain

  • Facilitates the interaction between USP10 (a deubiquitinating enzyme) and FASN

  • Promotes FASN deubiquitination, thereby increasing FASN protein stability
    This pathway represents a potential therapeutic target, as disrupting the SPICE1-FASN interaction could reduce OS cell proliferation.

How can the SPICE1-FASN interaction be experimentally disrupted and what are the implications?

The SPICE1-FASN interaction can be disrupted through several experimental approaches:

• Peptide inhibitors: Researchers have successfully developed a peptide (SPICE1 375-386) that disrupts the interaction between SPICE1 and FASN. This peptide effectively:

  • Reduces USP10 binding to FASN

  • Promotes FASN ubiquitination

  • Potentially inhibits OS cell proliferation

• Domain mutation: Targeted mutations in the CC1 domain of SPICE1 can disrupt its interaction with FASN. Experiments have shown that:

  • The CC1 domain of SPICE1 is essential for FASN binding

  • Cells reconstituted with SPICE1 lacking the CC1 domain show reduced interaction between USP10 and FASN

  • These cells also exhibit increased FASN ubiquitination levels

• Small molecule inhibitors: Rational drug design based on the structural interface between SPICE1 and FASN could yield small molecules that disrupt this interaction. Computer modeling using the HDOCK server has revealed that amino acids 377, 379, and 381 in the N-terminal region of SPICE1 are likely critical for its interaction with FASN .
  Disrupting this interaction has significant implications for cancer research and potential therapeutics, as it represents a novel approach for targeting OS and potentially other cancers where SPICE1 and FASN are dysregulated.

What techniques can be used to study SPICE1's effect on the ubiquitination pathway of FASN?

The following methodological approaches can be employed to investigate SPICE1's impact on FASN ubiquitination:

• Ubiquitination assays: Detect ubiquitinated FASN using immunoprecipitation followed by western blotting with anti-ubiquitin antibodies. Studies have shown that:

  • SPICE1 knockdown significantly increases FASN ubiquitination

  • SPICE1 overexpression decreases FASN ubiquitination levels

• Protein stability assays: Treat cells with cycloheximide (CHX) to inhibit protein synthesis and monitor FASN degradation over time. Research has demonstrated that:

  • SPICE1 knockdown accelerates FASN protein degradation

  • This effect is largely rescued by MG132 (proteasome inhibitor) but not by chloroquine (lysosome inhibitor)

  • This confirms that SPICE1 regulates FASN through the ubiquitin-proteasome pathway

• Deubiquitination studies: Investigate the role of USP10 in SPICE1-mediated FASN regulation through:

  • Co-transfection experiments with varying amounts of SPICE1 alongside USP10 and FASN

  • Co-immunoprecipitation to assess protein-protein interactions

  • Results have shown that SPICE1 overexpression progressively increases the interaction between USP10 and FASN

• Rescue experiments: Silence USP10 in cells overexpressing SPICE1 to determine if USP10 is necessary for SPICE1's effect on FASN ubiquitination. Research has confirmed that USP10 knockdown reverses the decrease in FASN ubiquitination caused by SPICE1 overexpression .
  These techniques provide complementary approaches to dissect the molecular mechanisms by which SPICE1 regulates FASN stability through the ubiquitin-proteasome system.

What are the key considerations when designing expression vectors for recombinant rat SPICE1?

When designing expression vectors for recombinant rat SPICE1, researchers should consider:

• Promoter selection: Choose appropriate promoters based on the expression system (bacterial, mammalian, etc.). For mammalian expression, CMV or EF1α promoters typically provide robust expression.

• Epitope tagging: Include epitope tags (FLAG, HA, Myc) to facilitate detection and purification. Position tags strategically to avoid interfering with functional domains. Studies have successfully used Flag-SPICE1 for co-immunoprecipitation experiments .

• Domain preservation: Ensure the CC1 domain (critical for FASN interaction) is preserved in the expression construct. Research has shown that this domain is essential for SPICE1's interaction with FASN and its oncogenic function .

• Codon optimization: Consider codon optimization for the expression system to enhance protein production. This is particularly important when expressing rat proteins in non-rodent expression systems.

• Inducible expression: For proteins that may affect cell division like SPICE1, consider using inducible expression systems (Tet-On/Off) to control expression timing and levels.

• Fusion proteins: For localization studies, GFP or other fluorescent protein fusions may be useful, but validate that the fusion does not disrupt localization or function.

How should researchers validate the functionality of recombinant rat SPICE1 in experimental systems?

Functional validation of recombinant rat SPICE1 should include multiple complementary approaches:

• Localization assessment: Confirm proper subcellular localization to centrioles, centrosomes, and spindles using immunofluorescence or live-cell imaging with appropriate markers. Co-localization experiments have been used to confirm the association of SPICE1 with FASN in OS cells .

• Interaction verification: Validate known protein-protein interactions (e.g., with FASN, USP10, CEP120) through co-immunoprecipitation or proximity ligation assays. Research has confirmed these interactions through co-immunoprecipitation followed by western blotting .

• Functional rescue: Perform rescue experiments in SPICE1-depleted cells to confirm that the recombinant protein restores normal function:

  • In proliferation assays (CCK8, colony formation, EdU)

  • In protein interaction studies (FASN binding, USP10 recruitment)

  • In ubiquitination regulation (FASN stability)

• Phenotypic analysis: Assess whether the recombinant protein produces expected phenotypes when overexpressed or introduced into knockout backgrounds:

  • Enhanced cell proliferation

  • Increased tumor growth in xenograft models

  • Stabilization of FASN protein levels

• Domain functionality: Validate the function of specific domains through mutational analysis or domain-deletion studies. The CC1 domain has been shown to be critical for SPICE1's interaction with FASN .

What are common challenges in producing soluble recombinant rat SPICE1 and how can they be addressed?

Producing soluble recombinant SPICE1 can be challenging due to its structural properties. Common issues and solutions include:

How can researchers address data inconsistencies when studying SPICE1 function across different experimental systems?

When facing inconsistent results across experimental systems when studying SPICE1, consider these methodological approaches:

• Standardize expression levels: Quantify SPICE1 expression across systems and adjust experimental conditions to achieve comparable levels. Western blotting and qRT-PCR have been used to confirm successful SPICE1 overexpression and knockdown in OS cells .

• Control for cell type-specific effects: SPICE1 function may vary across cell types. Use multiple cell lines and primary cells to establish consistent patterns. Studies have shown consistent effects of SPICE1 in both HOS and 143B osteosarcoma cell lines .

• Validate antibody specificity: Confirm antibody specificity using knockout/knockdown controls. Conduct side-by-side comparisons of different antibodies if available.

• Consider post-translational modifications: SPICE1 is phosphorylated by aurora kinase , and this modification may vary across systems. Use phospho-specific antibodies or phosphatase treatments to control for this variable.

• Account for interaction partners: The availability of interaction partners (FASN, USP10) may differ between systems. Quantify these proteins alongside SPICE1 to identify potential confounding factors.

• In vivo validation: Validate key findings in animal models to establish physiological relevance. Research has confirmed that SPICE1 knockdown inhibits tumor growth in nude mice, while SPICE1 overexpression accelerates tumor growth .
  By systematically addressing these factors, researchers can resolve inconsistencies and develop a more coherent understanding of SPICE1 function.

What emerging technologies might advance our understanding of SPICE1 function?

Several cutting-edge technologies hold promise for deepening our understanding of SPICE1:

• Cryo-electron microscopy: Determining the high-resolution structure of SPICE1 and its complexes would provide crucial insights into its functional mechanisms and potential for targeted therapeutics.

• Proximity labeling proteomics (BioID, APEX): These techniques could identify the complete interactome of SPICE1 in different cellular contexts, potentially revealing new interaction partners beyond FASN and USP10 .

• Single-cell proteomics: Examining SPICE1 expression and its effects at the single-cell level would reveal cell-to-cell variability and potentially identify distinct cellular subpopulations with differential SPICE1 activity.

• CRISPR screens: Genome-wide CRISPR screens in SPICE1-overexpressing or SPICE1-knockout backgrounds could identify synthetic lethal interactions and new functional pathways.

• Intravital imaging: Visualizing SPICE1 dynamics in live animals using fluorescent tags and intravital microscopy would provide insights into its behavior in intact tissues, particularly in tumor microenvironments.

• Protein degradation technologies (PROTACs): Developing SPICE1-targeting PROTACs could provide new tools for studying SPICE1 function through rapid and reversible protein depletion.
  These technologies would complement the established methodologies used in current SPICE1 research and potentially reveal new therapeutic opportunities.

What are the potential therapeutic implications of targeting the SPICE1/USP10/FASN signaling axis?

The SPICE1/USP10/FASN signaling axis represents a promising therapeutic target, particularly for osteosarcoma. Key considerations include:

• Peptide-based therapeutics: The peptide derived from SPICE1 amino acids 375-386 has shown efficacy in disrupting the SPICE1-FASN interaction . Further development could yield peptide therapeutics or peptidomimetics with improved pharmacological properties.

• Small molecule inhibitors: Rational drug design targeting the SPICE1-FASN interface, particularly focusing on the critical amino acids 377, 379, and 381 in SPICE1, could yield effective small molecule inhibitors .

• USP10 inhibitors: Targeting the deubiquitinating enzyme USP10 represents an alternative approach to disrupt this pathway. Research has shown that USP10 knockdown reverses the effects of SPICE1 overexpression on FASN ubiquitination .

• Combination therapies: Targeting the SPICE1/USP10/FASN axis in combination with conventional chemotherapeutics may enhance treatment efficacy for osteosarcoma patients.

• Biomarker development: SPICE1 overexpression correlates with poor prognosis in OS patients , suggesting its potential as a prognostic biomarker to guide treatment decisions. Future research should focus on developing specific inhibitors that disrupt this pathway, potentially leading to improved clinical outcomes for patients with osteosarcoma and potentially other cancers where this signaling axis is dysregulated.