Recombinant Gorilla gorilla gorilla SCAN domain-containing protein 1 (SCAND1)

What is SCAND1 and what are its primary functional characteristics?

SCAND1 is a SCAN domain-only protein that belongs to the SCAN domain-containing transcription factor (SCAN-TF) family. Unlike most members of this family that contain zinc finger domains (SCAN-ZF factors), SCAND1 is among the six zinc fingerless SCAN domain-only proteins identified in humans . The SCAN domain is a leucine-rich oligomerization domain that is highly conserved among the SCAN-TF family, which includes 64 members in humans .

SCAND1 functions primarily through hetero-oligomerization with other SCAN-ZF proteins, such as Myeloid Zinc Finger 1 (MZF1), via inter-SCAN domain interactions. This interaction transforms their roles to form a transcriptional repressor complex . Research has shown that SCAND1 expression correlates with maintaining epithelial features in cells, while its loss is associated with mesenchymal phenotypes of tumor cells . The protein plays a significant role in reversing the epithelial-to-mesenchymal transition (EMT), particularly in cancer contexts.

How is recombinant Gorilla gorilla gorilla SCAND1 expressed and purified for research purposes?

Recombinant Gorilla gorilla gorilla SCAND1 can be produced using standard molecular cloning techniques. Based on established protocols for SCAND1 expression, researchers typically begin with cDNA isolation or synthetic gene design based on the Gorilla gorilla gorilla SCAND1 sequence. The gene is then cloned into expression vectors such as pcDNA3 or pCMV6 with appropriate tags (e.g., Flag or myc) for detection and purification .

For expression, mammalian cell lines (such as HEK293T) or bacterial systems (E. coli) can be used depending on the research requirements. When using mammalian expression systems, transfection is typically performed using reagents like Lipofectamine 2000, followed by selection with antibiotics such as geneticin (0.2-1.6 μg/mL) for 2 weeks to establish stable clones . Purification is subsequently performed using affinity chromatography based on the tag used (e.g., anti-Flag affinity columns), followed by size exclusion chromatography to enhance purity.

Quality control measures should include SDS-PAGE analysis, Western blotting with antibodies against SCAND1 (such as ab64828 from Abcam) or tag-specific antibodies, and mass spectrometry to confirm protein identity and purity .

What experimental models are most suitable for studying SCAND1 function?

Research on SCAND1 has employed several experimental models that would be appropriate for studying the gorilla homolog:

• Cell culture models: Human prostate cancer cell lines such as DU-145 have been effectively used to study SCAND1 function . These models are particularly useful for investigating SCAND1's role in EMT, cell proliferation, and migration. Researchers can establish stable cell lines overexpressing SCAND1 using geneticin selection .

• Xenograft models: Mouse tumor xenograft models have been employed to study the in vivo effects of SCAND1 overexpression on tumor growth, proliferation (Ki-67 expression), mesenchymal characteristics (Vimentin expression), and metastasis .

• Co-expression systems: To study the interaction between SCAND1 and other proteins (e.g., MZF1, HP1γ), co-expression systems can be established by transfecting cells with multiple constructs .

• Reporter gene assays: These can be used to study the transcriptional repression activity of SCAND1 on specific target genes.

Each model provides distinct advantages for investigating different aspects of SCAND1 biology, from molecular interactions to physiological effects in complex tissue environments.

How does SCAND1 reverse epithelial-to-mesenchymal transition (EMT) in cancer cells?

SCAND1 has been demonstrated to reverse cancer cell mesenchymal and hybrid epithelial/mesenchymal (E/M) phenotypes to a more epithelial, less invasive status. This process involves several molecular mechanisms:

• E-cadherin and β-catenin relocation: SCAND1 overexpression leads to the relocation of E-cadherin and β-catenin, key proteins in maintaining epithelial cell-cell contacts .

• Transcriptional repression of EMT drivers: SCAND1 negatively correlates with EMT driver genes, including CTNNB1, ZEB1, ZEB2, and TGFBRs. This suggests that SCAND1 may directly or indirectly repress the expression of these genes .

• Coordination with MZF1: SCAND1 and MZF1 are mutually inducible and coordinately included in chromatin with heterochromatin protein HP1γ. This interaction forms a transcriptional repressor complex that may regulate EMT-related genes .

• Suppression of the MAP3K-MEK-ERK signaling pathway: SCAND1 overexpression suppresses tumor cell proliferation by reducing signaling through this pathway, which is important for mesenchymal characteristics and proliferation .

The net effect of these mechanisms is a transition from mesenchymal or hybrid E/M states back to a more epithelial phenotype, with consequent reductions in invasiveness, migration potential, and metastatic capability.

What are the molecular interactions between SCAND1 and MZF1, and how do they affect transcriptional regulation?

SCAND1 and MZF1 interact through their SCAN domains, forming heterodimers that alter their transcriptional regulatory functions. The molecular basis and consequences of this interaction include:

• Hetero-oligomerization mechanism: The SCAN domain of SCAND1 directly interacts with the SCAN domain of MZF1, forming a heterodimer that modifies the transcriptional activity of MZF1 .

• Mutual induction: SCAND1 and MZF1 are mutually inducible, creating a positive feedback loop that enhances their coordinated activity .

• Chromatin association: SCAND1 and MZF1 coordinately associate with chromatin through interaction with heterochromatin protein HP1γ (also known as CBX3) .

• Transcriptional repression: The SCAND1-MZF1 complex functions primarily as a transcriptional repressor. Hetero-oligomerization between SCAN domain-only molecules like SCAND1 and SCAN-ZF molecules like MZF1 transforms their roles to form a repressor complex .

• Target gene regulation: Co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression negatively correlates with EMT driver genes in prostate adenocarcinoma specimens .

This interaction represents a key mechanism by which SCAND1 exerts its effects on gene expression and cellular phenotype, particularly in the context of epithelial-mesenchymal transitions.

Researchers can employ several strategies to manipulate SCAND1 expression:

• Overexpression systems: Utilizing mammalian expression vectors such as pCMV6/SCAND1 with myc-Flag tags allows for stable or transient overexpression in cell lines. Selection with geneticin (0.2-1.6 μg/mL) can establish stable clones expressing SCAND1 .

• RNA interference: siRNA or shRNA targeting SCAND1 can be used to knockdown endogenous expression for loss-of-function studies.

• CRISPR/Cas9 gene editing: This approach can generate SCAND1 knockout cell lines or animal models for comprehensive functional analysis.

• Inducible expression systems: Tet-On or Tet-Off systems allow for temporal control of SCAND1 expression, facilitating studies of acute versus chronic effects.

• Domain-specific mutations: Introducing mutations in specific regions of SCAND1 can help dissect the functional importance of different protein domains.

For protein detection and functional analysis, researchers can use antibodies against SCAND1 (e.g., ab64828 from Abcam) or epitope tags (e.g., Flag-tag antibody M2 from Sigma) in Western blotting, immunoprecipitation, or immunofluorescence microscopy .

What are the known structural differences between human and Gorilla gorilla gorilla SCAND1, and how might they affect function?

While the search results do not provide specific information about structural differences between human and Gorilla gorilla gorilla SCAND1, the high conservation of SCAN domains across species suggests similar core functions. The SCAN domain is a leucine-rich oligomerization domain highly conserved among the SCAN-TF family members .

Potential structural differences might exist in:

• Amino acid sequence variations: Even with high conservation, small differences in amino acid sequences could affect protein-protein interaction affinities or specificities.

• Post-translational modification sites: Differences in phosphorylation, acetylation, or other modification sites could affect regulation and function.

• Protein stability: Variations in amino acid composition might affect protein half-life or susceptibility to degradation pathways.

Researchers interested in species-specific differences should consider:

• Performing sequence alignments between human and gorilla SCAND1

• Expressing both proteins recombinantly for comparative biochemical studies

• Using crystal structure analysis or protein modeling to identify structural variations

• Conducting comparative interaction studies with common binding partners like MZF1

These approaches would help elucidate any functional differences between the human and gorilla orthologs that might be relevant for experimental design or interpretation.

How can recombinant SCAND1 be used as a tool for cancer research?

Recombinant SCAND1 offers several applications in cancer research:

• EMT reversal studies: Recombinant SCAND1 can be used to investigate the molecular mechanisms of EMT reversal, which is critical for understanding metastasis inhibition. In mouse tumor xenograft models, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer .

• Transcriptional regulatory network analysis: As SCAND1 forms heterodimers with SCAN-ZF proteins like MZF1, recombinant SCAND1 can be used in chromatin immunoprecipitation (ChIP) assays to identify target genes regulated by these complexes .

• Therapeutic target validation: Since high expression of SCAND1 correlates with better prognosis in certain cancers, recombinant SCAND1 can be used to validate its potential as a therapeutic target .

• Biomarker development: Understanding the relationship between SCAND1 expression and cancer prognosis could lead to the development of new prognostic biomarkers. Kaplan-Meier analysis has shown that high expression of SCAND1 and MZF1 correlates with better prognoses in pancreatic cancer and head and neck cancers .

• Drug screening platforms: Cell-based assays using recombinant SCAND1 expression systems can be developed to screen for compounds that enhance SCAND1 expression or activity.

These applications highlight the versatility of recombinant SCAND1 as a research tool in understanding and potentially treating cancer through EMT modulation.

What challenges exist in using recombinant SCAND1 in experimental settings?

Researchers face several challenges when working with recombinant SCAND1:

• Protein solubility and stability: SCAN domain-containing proteins may have solubility issues when expressed recombinantly, requiring optimization of expression conditions and buffer compositions.

• Functional preservation: Ensuring that recombinant SCAND1 retains its native interaction capabilities with partners like MZF1 and HP1γ is crucial for valid experimental outcomes .

• Species-specific differences: When using Gorilla gorilla gorilla SCAND1 in human cell models, potential functional differences must be considered and controlled for.

• Context-dependent effects: SCAND1's effects appear to be context-dependent, with high expression correlating with better prognosis in some cancers (pancreatic, head and neck) but poorer prognosis in others (kidney) . This complexity necessitates careful experimental design and interpretation.

• Delivery methods: For in vivo studies, efficient delivery of recombinant SCAND1 or SCAND1-expressing constructs to target tissues presents a significant challenge.

• Quantification of protein-protein interactions: Accurately measuring the interaction dynamics between SCAND1 and its binding partners requires sophisticated biophysical techniques.

Addressing these challenges requires careful optimization of expression systems, purification protocols, and experimental conditions to ensure reliable and reproducible results.

How does SCAND1 expression correlate with prognosis in different cancer types?

SCAND1 expression shows varying correlations with prognosis across different cancer types:

These differential effects across cancer types highlight the context-dependent nature of SCAND1 function. The mechanisms underlying these different prognostic correlations may involve tissue-specific interaction partners, signaling pathway variations, or differences in the tumor microenvironment.

Co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression negatively correlates with EMT driver genes in prostate adenocarcinoma specimens , suggesting a common mechanism in cancers where SCAND1 is associated with better outcomes. Understanding these tissue-specific differences is critical for developing targeted therapeutic approaches based on SCAND1 modulation.

What are the optimal approaches for detecting SCAND1 expression and localization in experimental models?

Detecting SCAND1 expression and localization requires specific techniques optimized for this protein:

• Western blotting: For protein expression quantification, use antibodies against SCAND1 (e.g., ab64828 from Abcam) or epitope tags (e.g., Flag-tag antibody M2 from Sigma) if using tagged recombinant constructs . Chemiluminescence detection provides sensitive visualization of protein bands.

• Immunofluorescence microscopy: For cellular localization studies, fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and stain with anti-SCAND1 antibodies followed by fluorophore-conjugated secondary antibodies. Co-staining with DAPI (nuclear marker) and markers for specific cellular compartments can provide detailed localization information.

• Chromatin immunoprecipitation (ChIP): To study SCAND1's association with chromatin and its genomic binding sites, ChIP assays can be performed using anti-SCAND1 antibodies or epitope tag antibodies for tagged versions .

• Proximity ligation assay (PLA): This technique is valuable for visualizing and quantifying SCAND1's interactions with partners like MZF1 and HP1γ in situ with subcellular resolution.

• Flow cytometry: For quantitative analysis of SCAND1 expression in cell populations, particularly in heterogeneous samples, intracellular staining protocols can be adapted using anti-SCAND1 antibodies.

Each method offers distinct advantages, and researchers should select based on their specific experimental questions and available resources.

How can researchers effectively study SCAND1 interactions with other proteins in the SCAN-ZF family?

To study SCAND1 interactions with other SCAN-ZF family proteins, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP): This technique can identify protein-protein interactions between SCAND1 and potential partners. Using antibodies against SCAND1 or epitope tags, researchers can pull down SCAND1 complexes and analyze co-precipitated proteins by Western blotting .

• Bimolecular Fluorescence Complementation (BiFC): By fusing SCAND1 and potential interaction partners (e.g., MZF1) to complementary fragments of a fluorescent protein, researchers can visualize interactions in living cells when the proteins come together.

• Yeast two-hybrid screening: This approach can identify novel SCAN-ZF family members that interact with SCAND1.

• Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC): These biophysical techniques provide quantitative measurements of binding affinities and kinetics between purified SCAND1 and SCAN-ZF proteins.

• ChIP-sequencing: This technique can identify genomic regions where SCAND1 and its SCAN-ZF partners co-localize, providing insights into their collaborative gene regulatory functions .

• Mass spectrometry-based interactomics: Immunoprecipitation of SCAND1 followed by mass spectrometry analysis can identify novel interaction partners and post-translational modifications that may regulate these interactions.

These approaches provide complementary information about SCAND1's protein interaction network, helping to elucidate its role in transcriptional regulation and cellular function.

What gene expression analysis tools are most appropriate for studying SCAND1-mediated transcriptional changes?

Several gene expression analysis tools are particularly suitable for studying SCAND1-mediated transcriptional changes:

• RNA sequencing (RNA-seq): This comprehensive approach allows genome-wide profiling of transcriptional changes induced by SCAND1 overexpression or knockdown. It can identify both direct and indirect targets of SCAND1-mediated regulation.

• GO term enrichment analysis: Tools like GOrilla can identify enriched Gene Ontology terms in gene lists affected by SCAND1 manipulation, providing insights into biological processes, molecular functions, and cellular components modulated by SCAND1 .

• ChIP-seq followed by motif analysis: This approach can identify direct binding sites of SCAND1-containing complexes and associated DNA motifs, particularly when studying SCAND1's interaction with SCAN-ZF proteins like MZF1 .

• Quantitative RT-PCR: For targeted validation of specific genes identified in broader screens, qRT-PCR provides a sensitive and quantitative approach to confirm SCAND1-mediated gene expression changes.

• Promoter-reporter assays: These assays can directly test the effect of SCAND1 on the transcriptional activity of specific gene promoters, helping to distinguish direct from indirect effects.

• TCGA data mining tools: As demonstrated in the research, analyzing correlations between SCAND1 expression and other genes in cancer datasets from The Cancer Genome Atlas can provide valuable insights into SCAND1's regulatory networks in different cancer contexts .

GOrilla, in particular, offers advantages for analyzing ranked lists of genes, which is useful when studying gradual transcriptional changes induced by varying levels of SCAND1 expression. It employs a flexible threshold statistical approach and provides exact p-values for observed enrichments, along with an intuitive visualization of the results as a hierarchical structure .

What are the most promising therapeutic applications of SCAND1 research?

SCAND1 research shows several promising therapeutic applications:

• EMT reversal strategies: Given SCAND1's ability to reverse the epithelial-to-mesenchymal transition, developing approaches to enhance SCAND1 expression or activity could potentially reduce cancer invasiveness and metastasis .

• Biomarker development: SCAND1 expression levels could serve as prognostic biomarkers in specific cancer types. Kaplan-Meier analyses have already shown correlations between SCAND1 expression and prognosis in multiple cancer types .

• Targeted therapy development: Understanding the precise mechanisms by which SCAND1 suppresses the MAP3K-MEK-ERK signaling pathway could lead to novel targeted therapies that mimic or enhance this effect .

• Combination therapies: SCAND1-based approaches could potentially sensitize tumors to existing therapies by altering their epithelial/mesenchymal state, particularly in cancers with hybrid E/M phenotypes that are often associated with therapeutic resistance.

• Precision medicine applications: The differential effects of SCAND1 across cancer types (beneficial in some, potentially detrimental in others) highlight the importance of context-specific approaches, potentially leading to precision medicine strategies based on tumor-specific characteristics .

These applications represent promising directions for translating fundamental SCAND1 research into clinical benefits, particularly for cancers where EMT plays a significant role in progression and treatment resistance.

What unexplored aspects of SCAND1 biology warrant further investigation?

Several aspects of SCAND1 biology remain underexplored and warrant further investigation:

• Tissue-specific functions: The differential prognostic impact of SCAND1 across cancer types suggests tissue-specific functions that require further elucidation .

• Regulation of SCAND1 expression: The mechanisms controlling SCAND1 expression levels in different cellular contexts remain poorly understood.

• Post-translational modifications: How SCAND1 function is regulated by post-translational modifications such as phosphorylation, ubiquitination, or SUMOylation has not been extensively studied.

• Evolutionary conservation and divergence: Comparative studies of SCAND1 across species, including between humans and gorillas, could provide insights into evolutionarily conserved functions and species-specific adaptations.

• Role in normal development: While SCAND1's role in cancer has been explored, its functions in normal tissue development and homeostasis remain largely unknown.

• Interaction with non-SCAN proteins: Beyond its known interactions with SCAN-ZF proteins, SCAND1 may have additional protein partners that contribute to its cellular functions.

• Roles beyond transcriptional regulation: SCAND1 might have functions outside of its established role in transcriptional regulation that have yet to be discovered.

These unexplored areas represent exciting opportunities for researchers to contribute new knowledge to the field of SCAND1 biology and potentially uncover novel therapeutic targets or biomarkers.

How might advances in gene editing and protein engineering enhance SCAND1 research?

Advances in gene editing and protein engineering offer powerful approaches to enhance SCAND1 research:

• CRISPR/Cas9 genome editing: This technology enables precise modification of SCAND1 genomic loci to create knockout models, introduce specific mutations, or add reporter tags at endogenous loci. This allows for studying SCAND1 function in more physiologically relevant contexts than overexpression models.

• Domain-specific protein engineering: Creating chimeric proteins with specific domains of SCAND1 fused to other proteins can help dissect the functional importance of different regions, particularly the SCAN domain's interaction with various SCAN-ZF partners .

• Optogenetic and chemically inducible systems: These systems allow for temporal and spatial control of SCAND1 expression or activity, enabling studies of acute versus chronic effects and tissue-specific functions.

• Single-cell analysis technologies: Combined with genetic manipulation, these technologies can reveal cell-to-cell variability in SCAND1 expression and function, particularly in heterogeneous tumor samples.

• Protein structure determination: Advanced structural biology techniques can provide detailed insights into SCAND1's interaction interfaces with partners like MZF1 and HP1γ, potentially enabling the design of small molecules that modulate these interactions .

• In vivo gene editing: CRISPR-based approaches for in vivo modification of SCAND1 in animal models can provide insights into its systemic functions and therapeutic potential without the limitations of cell culture systems.

These technological advances offer unprecedented opportunities to understand SCAND1 biology at molecular, cellular, and organismal levels, potentially accelerating the translation of basic research findings into clinical applications.