BASP1 Human

What is BASP1 and why does it display unusual molecular weight patterns in Western blots?

BASP1 (Brain Acid Soluble Protein 1) functions as a transcriptional cosuppressor belonging to the BASP1 family of proteins. Despite having a predicted molecular weight of approximately 23 kDa, BASP1 exhibits unusual migration patterns in SDS-PAGE analyses . This occurs because BASP1 forms oligomers in SDS, resulting in multiple bands ranging from 30-150 kDa . The apparent molecular weight of BASP1 also varies depending on SDS concentration - appearing as 56 kDa in 8% SDS-PAGE and 41 kDa in 13% SDS-PAGE .

Human BASP1 exists in at least two forms: one approximately 48-52 kDa and another 32-40 kDa in size, which are not functionally equivalent . Furthermore, BASP1 can undergo SUMOylation, adding about 20 kDa to its apparent molecular weight . When conducting Western blots for BASP1, researchers should anticipate multiple bands and consider using positive controls with known BASP1 expression patterns such as HeLa or DU145 cell lines, which show specific bands at approximately 60 kDa as well as lower molecular weight isoforms .

Which methodological approaches are most reliable for detecting and studying BASP1 in experimental settings?

For reliable detection of BASP1 in experimental settings, researchers should employ a combination of complementary techniques:

Western Blotting Protocol:

• Use specific antibodies such as Human BASP1 Antigen Affinity-purified Polyclonal Antibody (e.g., R&D Systems AF6479)

• Conduct blotting under reducing conditions on 4-20% SDS-PAGE gels

• Expect multiple bands, including a specific band at approximately 60 kDa and additional lower molecular weight isoforms

• Normalize using β-actin (1:10,000, ab213262 from Abcam) as an internal reference

RT-qPCR Approach:

• Use validated primers for human BASP1:

  • Forward: 5′-GCAACTCGTTTGCAGCGG-3′

  • Reverse: 5′-CCCATCTTGGAGTTCTCGGC-3′

• For mouse studies, different primers are required:

  • Forward: 5′-GAGAGCCTTTGCTGAGCGAC-3′

Immunofluorescence Applications:

• This technique effectively visualizes BASP1 subcellular localization, particularly important for studying its nuclear functions in transcriptional regulation

• Should be performed with appropriate controls to verify specificity

Protein levels should be quantified according to internal reference band density using appropriate software such as ImageLab for accurate comparative analyses .

How does BASP1 function as a transcriptional corepressor in cellular contexts?

BASP1 functions as a transcriptional corepressor primarily through its association with Wilms tumor 1 (WT1), converting WT1 from a transcriptional activator to a repressor . This mechanism involves several key components and processes:

Cholesterol-Dependent Repression:

• BASP1 directly interacts with cholesterol within the cell nucleus through a conserved cholesterol interaction motif

• BASP1 actively recruits cholesterol to the promoter regions of WT1 target genes

• Mutation of BASP1 to prevent cholesterol interaction or treatment with cholesterol biosynthesis inhibitors blocks BASP1's transcriptional repressor function

Chromatin Remodeling Mechanism:

• The BASP1-cholesterol interaction is required for BASP1-dependent chromatin remodeling

• This interaction affects nucleosome organization and accessibility at target genes

• The process represents a direct role for cholesterol in transcriptional regulation through structural changes to chromatin

Pathway Regulation:

• BASP1 inhibits the mRNA and protein expression of WT1, Wnt, and β-catenin in cancer cells

• This inhibition creates regulatory networks that control gene expression programs involved in cell differentiation, proliferation, and apoptosis

This mechanism connects lipid metabolism to transcriptional control, suggesting that alterations in cholesterol levels or trafficking could affect gene expression programs regulated by BASP1 .

What is the molecular relationship between BASP1 and cholesterol in transcriptional regulation?

BASP1 and cholesterol have a direct functional relationship in gene regulation, representing a novel mechanism for nuclear cholesterol function:

Direct Molecular Interaction:

• BASP1 directly binds to cholesterol in the cell nucleus through a specific and conserved cholesterol interaction motif

• This interaction is essential for BASP1's function as a transcriptional repressor

Gene-Specific Targeting:

• BASP1 actively recruits cholesterol to promoter regions of specific target genes

• This recruitment provides a mechanism for targeted cholesterol function in transcription regulation

• The specificity allows for gene-selective repression rather than global transcriptional effects

Functional Requirement:

• The BASP1-cholesterol interaction is mandatory for transcriptional repression

• Mutations preventing BASP1-cholesterol binding or treatments reducing cellular cholesterol inhibit BASP1's repressor function

• This demonstrates that cholesterol is functionally required for BASP1 activity, not merely associated with it

This relationship provides the first clear evidence for a direct role of cholesterol in transcriptional regulation, connecting cellular metabolism to gene expression control . This mechanism may be particularly relevant in conditions with disrupted cholesterol metabolism, such as cardiovascular disease or certain metabolic disorders.

Through which signaling pathways does BASP1 influence cell growth and metastasis in cancer models?

BASP1 regulates cell growth and metastasis through several interconnected signaling pathways, particularly in cancer contexts:

WT1 Regulatory Axis:

• BASP1 inhibits the transcriptional activation of WT1, which functions as an oncogene in multiple cancers

• By suppressing WT1, BASP1 indirectly affects numerous downstream targets involved in cell proliferation and survival

Wnt/β-catenin Pathway Modulation:

• BASP1 overexpression significantly inhibits both mRNA and protein expression of key Wnt/β-catenin pathway components, including Wnt and β-catenin themselves

• This inhibition suppresses the pathway at a transcriptional level, creating a negative regulatory effect

• The inhibition sequence appears to function as: BASP1 → WT1 → Wnt/β-catenin

Apoptotic Pathway Regulation:

• BASP1 overexpression promotes the expression of pro-apoptotic proteins like Bax and caspase-3

• It simultaneously inhibits anti-apoptotic proteins like Bcl-2

• This dual regulation enhances cancer cell apoptosis, contributing to tumor suppression

Metastasis-Related Pathways:

• BASP1 suppresses matrix metalloproteinases MMP-2 and MMP-9, which are critical for invasion and metastasis

• This inhibition reduces the invasive and metastatic potential of cancer cells

EGFR Pathway in Endothelial Context:

• In endothelial cells, BASP1 positively regulates the Epidermal Growth Factor Receptor (EGFR) pathway

• This regulation promotes endothelial cell apoptosis under high glucose conditions

These pathway interactions explain BASP1's observed effects on cell proliferation, migration, invasion, and apoptosis across multiple cancer models and endothelial dysfunction contexts .

How does BASP1 contribute to endothelial dysfunction in diabetes and cardiovascular complications?

BASP1 plays a significant role in diabetes-related endothelial dysfunction, contributing to vascular complications in diabetic patients:

Pathological Upregulation:

• Bioinformatics analyses of databases related to diabetes with coronary heart disease identified BASP1 as a significantly upregulated gene

• High glucose conditions induce BASP1 upregulation in endothelial cells in a time-dependent manner

• This upregulation appears specific to diabetes-related cardiovascular pathology

Endothelial Injury Mechanism:

• BASP1 actively promotes endothelial cell injury under high glucose conditions

• Silencing BASP1 expression alleviates damage caused by high glucose to endothelial cells, demonstrating a causal relationship

• BASP1-related injury manifests through multiple endothelial dysfunctions

EGFR Pathway Activation:

• BASP1 positively regulates the Epidermal Growth Factor Receptor (EGFR) pathway in endothelial cells

• The promoting effect of BASP1 on endothelial cell apoptosis is achieved through activation of the EGFR pathway

• EGFR inhibitors (such as gefitinib) can counteract BASP1's deleterious effects, confirming this mechanistic pathway

Comprehensive Endothelial Impact:

• High BASP1 expression negatively affects multiple essential endothelial functions:

  • Increases apoptosis rates

  • Reduces cellular migration capability

  • Impairs tube formation (angiogenesis)

  • Enhances inflammatory responses

  • Increases reactive oxygen species (ROS) production

These findings establish BASP1 as a potential therapeutic target for diabetes complicated with cardiovascular disease, as it appears to be a critical mediator in the endothelial dysfunction that contributes to diabetic vascular complications .

What explains the apparently contradictory roles of BASP1 across different cancer types?

BASP1 exhibits variable expression patterns and seemingly contradictory functions across different cancer types, a complexity explained by several factors:

Differential Expression Patterns:

Context-Dependent Mechanisms:

• Epigenetic Regulation: In some cancers, BASP1 downregulation is linked to aberrant promoter methylation

• Tissue-Specific Partners: BASP1's interaction with partner proteins like WT1 varies across tissue types

• Pathway Dependency: The baseline activity and importance of pathways regulated by BASP1 (Wnt/β-catenin, EGFR) differ across cancer types

• Post-Translational Modifications: Various modifications including SUMOylation may change BASP1 function across contexts

Functional Effects in Cancer Models:

• In gastric cancer: BASP1 overexpression suppresses proliferation, migration, invasion and promotes apoptosis

• In thyroid cancer: BASP1 inhibits cell proliferation and migration while promoting apoptosis

• In acute myeloid leukemia: BASP1 represses growth by inhibiting proliferation and promoting apoptosis

This context-dependent behavior suggests BASP1's function is highly dependent on the cellular environment, including tissue type, genetic background, and the status of interacting pathways . These variations explain why BASP1 can act as a tumor suppressor in many cancers while potentially contributing to progression in others.

What experimental design considerations are critical when studying BASP1-cholesterol interactions?

Studying BASP1-cholesterol interactions requires careful experimental design with attention to several critical parameters:

Protein Preparation Protocol:

• Use E. coli-derived recombinant human BASP1 (Gly2-Ala45) for consistency

• Store at -20 to -70°C for up to 12 months from receipt

• After reconstitution, store at 2-8°C under sterile conditions for up to 1 month, or at -20 to -70°C for up to 6 months

• Avoid repeated freeze-thaw cycles by using a manual defrost freezer

Cholesterol Manipulation Strategies:

• Include experimental conditions with cholesterol biosynthesis inhibitors (statins) to test functional dependency

• Use cholesterol-depleting agents (methyl-β-cyclodextrin) to remove cellular cholesterol

• Include controls with mutated cholesterol-binding motifs in BASP1 to confirm specificity

Interaction Detection Methods:

• For direct binding assessment, employ surface plasmon resonance or isothermal titration calorimetry

• In cellular studies, use chromatin immunoprecipitation (ChIP) assays to demonstrate BASP1 and cholesterol co-localization at specific promoters

• Sequential ChIP can determine if BASP1 and WT1 simultaneously occupy the same promoter regions

Functional Validation Approaches:

• Reporter gene assays with WT1 target promoters can measure functional outcomes of the interaction

• Gene expression analysis following BASP1 mutation or cholesterol depletion can identify affected pathways

• Chromatin accessibility assays can determine if the BASP1-cholesterol interaction affects chromatin structure

Data Analysis Considerations:

• Statistical analysis should employ appropriate software (e.g., GraphPad Prism)

• Use t-tests for two-group comparisons and ANOVA with post-hoc tests for multiple groups

• Set significance thresholds at p < 0.05

These experimental design considerations ensure robust analysis of BASP1-cholesterol interactions and their functional significance in transcriptional regulation .

What methodological approaches enable effective manipulation of BASP1 expression in cell culture systems?

Effective manipulation of BASP1 expression in cell culture models requires careful consideration of multiple technical approaches:

Overexpression Methodologies:

• Vector Selection: pcDNA3.1 vectors containing the BASP1 coding sequence have been successfully used for overexpression studies

• Cell Line Selection: AGS and HGC-27 gastric cancer cells, HUVECs, and primary mouse aortic endothelial cells demonstrate good transfection efficiency

• Control Implementation: Include empty vector controls (e.g., pcDNA3.1-NC) to account for transfection effects independent of BASP1 expression

• Expression Verification: Confirm overexpression by Western blot and RT-qPCR, noting that BASP1 will show multiple bands due to its unusual migration patterns

Knockdown Strategies:

• siRNA Approach: Effective siRNAs targeting BASP1 have been successfully employed in endothelial cells and cancer cell lines

• Validation Methods: Confirm knockdown at both protein and mRNA levels using Western blot and RT-qPCR

• Control Implementation: Include non-targeting siRNA controls to account for non-specific effects

Experimental Model Systems:

• Cancer Models: HeLa (cervical), DU145 (prostate), AGS and HGC-27 (gastric) cell lines have been validated for BASP1 studies

• Endothelial Models: HUVECs and primary mouse aortic endothelial cells are appropriate for studying BASP1's role in endothelial function

• Environmental Conditions: For endothelial studies, high glucose treatment (typically 25-30 mM) can be used to induce BASP1 upregulation in a time-dependent manner

Functional Readout Assays:

• Proliferation Assessment: Colony formation assays and EdU incorporation assays effectively measure BASP1's impact on cell proliferation

• Apoptosis Quantification: Flow cytometry for apoptosis detection, coupled with Western blot analysis of apoptosis-related proteins (Bax, caspase-3, Bcl-2)

• Migration/Invasion Analysis: Transwell assays with or without Matrigel coating, with complementary Western blot analysis of MMPs

• Pathway Analysis: Western blot and RT-qPCR analysis of pathway components (WT1, Wnt, β-catenin) to assess mechanistic effects

These methodological approaches provide a comprehensive framework for manipulating and studying BASP1's function in relevant cell culture systems .

What design principles should guide primer development for accurate BASP1 detection in qPCR?

Designing effective primers for BASP1 detection in qPCR requires careful attention to several technical considerations:

Validated Primer Sequences:

• Human BASP1:

  • Forward: 5′-GCAACTCGTTTGCAGCGG-3′

  • Reverse: 5′-CCCATCTTGGAGTTCTCGGC-3′

• Mouse BASP1:

  • Forward: 5′-GAGAGCCTTTGCTGAGCGAC-3′

Target Region Selection Principles:

• Select regions conserved across known BASP1 transcript variants to detect all isoforms

• Consider targeting exon-exon junctions to minimize genomic DNA amplification

• Avoid regions with known polymorphisms that could affect primer binding

• Check for potential cross-reactivity with related genes

Primer Design Parameters:

• Optimal primer length: 18-25 nucleotides (as seen in the validated primers above)

• GC content: 40-60% (the validated primers follow this guideline)

• Melting temperature (Tm): 58-62°C with minimal difference between forward and reverse primers

• Avoid secondary structures, self-complementarity, and complementarity between primer pairs

• Design amplicons of 70-150 bp for efficient amplification and better qPCR performance

Reference Gene Selection:

• β-actin has been successfully used as a reference gene with the following primers:

  • Human β-actin forward: 5′-GCACAGAGCCTCGCCTTT-3′

  • Human β-actin reverse: 5′-CACAGGACTCCATGCCCAG-3′

• Validate reference gene stability under your specific experimental conditions

Validation and Controls:

• Validate primer efficiency using standard curves with serial dilutions of template

• Confirm amplicon specificity through melt curve analysis and/or gel electrophoresis

• Include no-template controls and, when possible, no-reverse-transcriptase controls

Following these design principles ensures reliable and reproducible qPCR results for BASP1 detection across different experimental conditions and cell types .

How do post-translational modifications explain BASP1's diverse functional roles?

Post-translational modifications (PTMs) of BASP1 likely serve as key molecular switches that diversify its function across different cellular contexts:

SUMOylation Regulation:

• BASP1 undergoes SUMOylation, adding approximately 20 kDa to its apparent molecular weight

• This modification potentially alters BASP1's interaction capabilities, subcellular localization, or stability

• SUMOylation often regulates transcription factor activity, suggesting it may modulate BASP1's function as a transcriptional corepressor

• Different cellular contexts may exhibit variable SUMOylation machinery activity, potentially explaining tissue-specific functions

Lipidation Mechanisms:

• BASP1 is described as a lipidated WT1 transcriptional corepressor

• It interacts with cholesterol through a conserved cholesterol interaction motif

• Variations in cellular lipid composition or metabolism affect the extent and type of BASP1 lipidation

• This lipidation-dependent functionality connects BASP1 to cellular metabolic state, explaining its role in conditions like diabetes

Potential Phosphorylation:

• While not explicitly confirmed in the search results, BASP1 contains potential phosphorylation sites

• Different signaling cascades active in various cellular contexts could result in differential phosphorylation patterns

• This could connect BASP1 function to specific signaling pathways active in different tissues or disease states

PTM Crosstalk Hypothesis:

• Multiple modifications likely occur simultaneously on BASP1, creating a complex "PTM code"

• The presence of one modification might influence the likelihood or effect of others

• This crosstalk could generate numerous functionally distinct forms of BASP1 across different tissues

Understanding how these modifications regulate BASP1 function could provide insights into its diverse roles and potentially explain seemingly contradictory effects in different cellular contexts . This knowledge might also reveal opportunities for targeted therapeutic interventions in diseases where BASP1 plays a role.

What explains the tissue-specific expression and function of BASP1 in human physiology?

Despite growing research on BASP1, several significant gaps limit our understanding of its tissue-specific functions:

Differential Expression Patterns:

• BASP1 shows variable expression across different human tissues according to the Human Protein Atlas

• The functional significance of these expression patterns remains largely uncharacterized

• Limited understanding exists regarding how BASP1 expression changes during development or in response to physiological and pathological stimuli

Isoform Distribution:

• At least two forms of BASP1 exist in humans (48-52 kDa and 32-40 kDa), which are not functionally equivalent

• The tissue distribution and relative abundance of these isoforms remain poorly characterized

• The functional differences between these isoforms and how they contribute to tissue-specific roles require further investigation

Regulatory Network Variations:

• The upstream regulators controlling BASP1 expression in different tissues remain largely unknown

• Tissue-specific transcription factors and epigenetic mechanisms likely regulate BASP1 expression differently across tissues

• Systemic factors (hormones, metabolic state) may influence BASP1 expression and function in tissue-specific ways

Variable Protein Interactions:

• While BASP1's interaction with WT1 and cholesterol has been established , its complete interactome likely varies across tissues

• Tissue-specific protein-protein interaction networks involving BASP1 remain unmapped

• These differential interactions likely contribute to tissue-specific functions but require further characterization

These knowledge gaps highlight the need for integrated approaches combining proteomics, transcriptomics, and functional studies across different tissues, alongside the development of better tools for BASP1 detection and characterization in tissue-specific contexts .