SNTA1 Human

What is SNTA1 and what are its primary functions in human cells?

SNTA1 (Alpha-1-syntrophin) is a scaffolding protein that orchestrates signal transduction complexes by clustering various signaling components. It belongs to the syntrophin family of proteins, which includes alpha-1, beta-1, beta-2, gamma-1, and gamma-2 syntrophins . SNTA1 primarily functions as a critical link between the extracellular matrix and intracellular cytoskeleton by connecting with the dystrophin-associated protein complex (DAPC) . It plays essential roles in:

• Activation of Rac1 signaling pathways

• Regulation of reactive oxygen species (ROS) generation

• Facilitation of cell migration processes

• Modulation of ion channel function, particularly cardiac sodium channels (Nav1.5)

• Proper subcellular localization and expression of membrane proteins

How is SNTA1 involved in protein complex formation?

SNTA1 participates in complex formation with multiple proteins to regulate cellular signaling. Research has identified a novel complex involving SNTA1, P66shc, and Grb2 proteins that is implicated in Rac1 activation . When SNTA1 and P66shc are overexpressed, they cause significant displacement of Sos1 protein from Grb2, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1 . This mechanism leads to Rac1 activation, which subsequently increases reactive oxygen species (ROS) production and enhances migratory potential in human cells, particularly cancer cells . Additionally, SNTA1 binds to the PDZ domain motif of Nav1.5 (a cardiac voltage-gated sodium channel) and interacts with neuronal nitric oxide synthase (nNOS) and plasma membrane Ca-ATPase subtype 4b (PMCA4b) .

What techniques are most effective for detecting SNTA1 protein expression in tissue samples?

For detecting SNTA1 protein expression in tissue samples, multiple complementary approaches should be employed:

• Western blotting: Provides quantitative assessment of SNTA1 protein levels. This technique has been successfully used to confirm SNTA1 knockout in gene-editing experiments and to compare expression levels across different tissues .

• Immunohistochemistry/Immunofluorescence: Allows visualization of SNTA1 spatial distribution within tissues and cells. Immunofluorescence has been used to confirm the expression of SNTA1 in various cell types, providing insights into its subcellular localization .

• Co-immunoprecipitation assays: Essential for studying SNTA1's interactions with partner proteins such as P66shc, Grb2, Nav1.5, nNOS, and PMCA4b .

• Tissue microarrays: Enables high-throughput comparative analysis of SNTA1 expression across multiple tissue samples simultaneously, as demonstrated in cancer tissue studies .

When analyzing expression in cancer tissues, comparative analysis with matched normal tissue is crucial for meaningful interpretation of results .

What are the most effective approaches for SNTA1 knockout in human cell models?

The CRISPR-Cas9 system has proven highly effective for SNTA1 knockout in human cell models. The methodological approach includes:

• sgRNA design: Target public exons close to the start codon. For example, research has successfully targeted exon 2 of SNTA1 with sgRNA (5′-attggcaggacag-3′) .

• Target site selection: The pleckstrin homology 1 (PH1) domain has been used as an effective target site for sgRNA .

• Verification of knockout:

  • DNA sequencing to confirm genetic modification

  • Western blotting to verify absence of protein expression

  • Synthego analysis of sequencing results to evaluate knockout efficiency

• Pluripotency maintenance: For stem cell models, it's essential to verify that the knockout cells maintain pluripotency markers (SSEA4, NANOG, SOX2, DPPA4, OCT-4) to ensure the knockout hasn't affected basic cellular properties .

The H9 embryonic stem cell line has been successfully used as a model for SNTA1 knockout, as exemplified by the establishment of the WAe009-A-50 cell line .

How can researchers effectively study SNTA1 protein interactions with its binding partners?

To effectively study SNTA1 protein interactions with binding partners, researchers should employ multiple complementary techniques:

• Co-immunoprecipitation (Co-IP): This gold-standard technique has successfully demonstrated SNTA1's interactions with P66shc, Grb2, nNOS, and PMCA4b. Using specific antibodies against SNTA1 or its potential binding partners, researchers can pull down protein complexes and analyze them to confirm interactions .

• GST-fusion protein pull-down assays: This approach has proven particularly valuable for SNTA1 research. For example, using a GST-fusion protein of the C terminus of SCN5A (sodium channel), researchers demonstrated that wild-type SNTA1 interacts with SCN5A, nNOS, and PMCA4b, while the A390V-SNTA1 mutation disrupted the association with PMCA4b .

• Proximity ligation assays: This technique can detect protein interactions in situ, providing spatial information about where in the cell these interactions occur.

• Heterologous expression systems: Expressing SNTA1 along with potential binding partners in cell lines (such as HEK293) allows for controlled studies of protein interactions and their functional consequences .

• Mutational analysis: Creating specific mutations in SNTA1 (such as the A390V mutation) can reveal which domains are critical for particular protein interactions .

When studying SNTA1 interactions, it's essential to consider the cellular context, as interactions may vary between different cell types and physiological conditions.

What are the challenges in generating stable SNTA1-deficient human cardiomyocyte models?

Generating stable SNTA1-deficient human cardiomyocyte models presents several methodological challenges:

• Source cell selection: Human embryonic stem cells (hESCs) like the H9 line are preferred starting points, but ethical considerations and specialized culture requirements add complexity .

• Efficient cardiac differentiation: After SNTA1 knockout, ensuring efficient differentiation into cardiomyocytes requires careful protocol optimization. The CardioEasy kit containing small molecule inhibitors has been successfully employed for this purpose .

• Phenotypic stability: SNTA1-deficient cardiomyocytes may develop compensatory mechanisms over time that mask the primary effects of SNTA1 deficiency.

• Functional assessment complexity: Comprehensive evaluation requires multiple techniques:

  • Electrophysiological studies for ion channel function

  • Calcium imaging for excitation-contraction coupling

  • Contractility measurements

  • Protein expression and localization analyses

• Heterogeneity in differentiated populations: Ensuring a pure population of cardiomyocytes with consistent SNTA1 deficiency can be challenging.

Researchers have overcome these challenges by establishing protocols that include rigorous validation of pluripotency after gene editing, carefully optimized cardiac differentiation methods, and comprehensive functional characterization of the resulting cardiomyocytes .

How does SNTA1 regulate cardiac sodium channel function?

SNTA1 regulates cardiac sodium channel (Nav1.5) function through multiple mechanisms:

This regulatory mechanism has significant implications for cardiac electrophysiology, as increased late sodium current is the characteristic biophysical dysfunction for sodium-channel-mediated Long QT Syndrome (LQT3) .

What is the evidence linking SNTA1 mutations to Long QT Syndrome?

The evidence linking SNTA1 mutations to Long QT Syndrome (LQTS) is substantial and multifaceted:

• Clinical case identification: A missense mutation (A390V-SNTA1) was identified in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms) who was negative for mutations in the 11 known LQTS-susceptibility genes .

• Molecular mechanism studies: The A390V mutation was shown to selectively disrupt the association of PMCA4b with the SNTA1-nNOS complex while maintaining interaction with SCN5A and nNOS .

• Functional consequences:

  • A390V-SNTA1 increased direct nitrosylation of SCN5A

  • When expressed with SCN5A, nNOS, and PMCA4b in heterologous cells, A390V-SNTA1 increased both peak and late sodium current compared to wild-type SNTA1

  • The increase in sodium current was partially inhibited by NOS blockers

  • Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current

• Pathophysiological consistency: The increased late sodium current observed with the A390V-SNTA1 mutation is characteristic of the biophysical dysfunction seen in sodium-channel-mediated LQTS (LQT3) .

These findings establish SNTA1 as a rare LQTS-susceptibility gene and highlight the SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current in the heart .

What phenotypes are observed in SNTA1-deficient human cardiomyocytes?

SNTA1-deficient human cardiomyocytes demonstrate several distinct phenotypes:

• Hypertrophic characteristics: SNTA1 knockout in human cardiomyocytes leads to cellular hypertrophy, consistent with observations in Snta1 knockout mice that showed left ventricular posterior wall thickening .

• Abnormal sodium channel function: Deficiency in SNTA1 affects the localization, expression, and function of Nav1.5 sodium channels, which can alter cardiomyocyte electrophysiology .

• Altered membrane channel regulation: SNTA1 is involved in the regulation of membrane volume on Kir2.1 and Kir2.2 channels, and its absence affects these regulatory mechanisms .

• Disrupted signaling between extracellular matrix and cytoskeleton: As SNTA1 connects the dystrophin-associated protein complex (DAPC) with the cytoskeleton, its absence disrupts this important signaling pathway .

• Abnormal myocardial performance: This is consistent with findings in Snta1 knockout mice that showed abnormal myocardial performance index .

These findings highlight the importance of using human cardiomyocytes derived from SNTA1-knockout embryonic stem cells to study the cellular phenotype, as they provide a more relevant model for human cardiac conditions compared to non-human cardiomyocytes used in previous research .

How does SNTA1 expression vary across different human cancer types?

SNTA1 expression shows significant tissue-specific variation across different human cancer types:

• Esophageal cancer:

  • Significantly decreased expression in both esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) compared to respective normal tissues

• Breast cancer:

  • Significantly increased expression compared to normal breast tissue

• Other cancer types:

  • No significant difference in expression was observed in stomach, lung, colon, and rectal cancers compared to their normal tissue counterparts

This differential expression pattern suggests that SNTA1 may play tissue-specific roles in carcinogenesis, potentially functioning as a tumor suppressor in esophageal tissues while contributing to oncogenesis in breast tissue . These findings highlight the importance of tissue context when studying SNTA1's role in cancer.

What are the mechanisms by which SNTA1 influences cancer cell migration and invasiveness?

SNTA1 influences cancer cell migration and invasiveness through several interconnected mechanisms:

• Rac1 activation pathway: SNTA1 forms a complex with P66shc and Grb2 proteins that is involved in activating Rac1, a small GTPase critical for cell migration. Overexpression of SNTA1 and P66shc significantly increases Rac1 activation .

• Displacement of signaling proteins: When SNTA1 and P66shc are overexpressed, they cause displacement of Sos1 protein from Grb2, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1, which further enhances Rac1 activation .

• Reactive oxygen species (ROS) production: SNTA1-mediated Rac1 activation results in increased ROS production, which can promote migratory behavior in cancer cells .

• Enhanced migratory potential: The combination of these mechanisms leads to increased migratory potential in human cancer cells, particularly in breast cancer where SNTA1 is overexpressed .

• Tissue-specific effects: The impact of SNTA1 on migration likely varies by tissue type, as suggested by its differential expression patterns across various cancers .

These findings indicate that targeting the SNTA1-P66shc-Grb2 complex might represent a potential therapeutic approach for reducing cancer cell migration and invasiveness, particularly in breast cancer where SNTA1 is overexpressed .

What experimental approaches are most effective for studying SNTA1's role in tumor progression?

For studying SNTA1's role in tumor progression, several complementary experimental approaches are particularly effective:

• Expression profiling in clinical samples:

  • Immunohistochemistry of tissue microarrays containing matched tumor and normal samples

  • Western blot analysis for quantitative comparison of protein levels

  • qRT-PCR for mRNA expression analysis

• Functional manipulation in cell lines:

  • siRNA and shRNA for SNTA1 downregulation to assess loss-of-function effects

  • Overexpression systems to evaluate gain-of-function effects

  • CRISPR-Cas9 for complete knockout studies

• Migration and invasion assays:

  • In vitro wound healing assays to assess cell migration

  • Transwell migration and invasion assays

  • 3D spheroid invasion assays for more physiologically relevant models

• Molecular mechanism studies:

  • Co-immunoprecipitation to identify SNTA1 interaction partners in cancer cells

  • Rac1 activation assays to measure effects on this key migration-related pathway

  • ROS generation assays to assess oxidative stress effects

• In vivo models:

  • Xenograft models using SNTA1-manipulated cancer cells

  • Patient-derived xenografts to maintain tumor heterogeneity

  • Metastasis models to specifically assess SNTA1's role in cancer spread

• Correlation with clinical outcomes:

  • Analysis of SNTA1 expression in relation to patient survival, tumor stage, and metastatic status

These approaches should be tailored to the specific cancer type being studied, considering the tissue-specific expression patterns of SNTA1 observed across different cancers .

How might post-translational modifications of SNTA1 affect its function in different cellular contexts?

Post-translational modifications (PTMs) of SNTA1 likely play crucial roles in regulating its function across different cellular contexts:

• Phosphorylation:

  • Potential phosphorylation sites on SNTA1 may regulate its binding affinity to partners such as nNOS, PMCA4b, and Nav1.5

  • Different kinase activities in various cell types could lead to tissue-specific SNTA1 function through differential phosphorylation patterns

  • Research methods should include phospho-specific antibodies, mass spectrometry, and phosphomimetic mutants to assess functional impacts

• S-nitrosylation:

  • While SNTA1 regulates S-nitrosylation of Nav1.5, SNTA1 itself might be subject to nitrosylation in a feedback mechanism

  • This modification could alter scaffold properties and protein-protein interactions

  • Biotin switch assays and mass spectrometry approaches would be valuable for investigating this possibility

• Ubiquitination and SUMOylation:

  • These modifications could regulate SNTA1 stability and turnover

  • They may be differentially regulated in disease states such as cancer or cardiac pathologies

  • Proteasome inhibitors and ubiquitin/SUMO-specific immunoprecipitation approaches would help elucidate these mechanisms

• Context-dependent modifications:

  • Cardiac stress conditions might trigger specific PTMs that alter SNTA1's interaction with ion channels

  • In cancer cells, oncogenic signaling might induce unique modification patterns that enhance migration-promoting functions

  • Comparative proteomic analysis across different tissues and disease states would provide valuable insights

Understanding these modifications could reveal how SNTA1 function is fine-tuned in different physiological and pathological contexts, potentially identifying novel therapeutic targets.

What are the contradictions in current SNTA1 research findings and how might they be resolved?

Several notable contradictions exist in current SNTA1 research findings:

• Opposing roles in different cancers:

  • SNTA1 appears downregulated in esophageal cancers but upregulated in breast cancer

  • Resolution approach: Tissue-specific transcriptional regulation studies and context-dependent protein interaction mapping could explain these differences

  • Methodological solution: Single-cell analysis to identify cell type-specific expression patterns within tumor microenvironments

• Cardiac phenotype variations:

  • Some studies suggest SNTA1 deficiency leads to cardiac hypertrophy, while others focus primarily on arrhythmia phenotypes

  • Resolution approach: Comprehensive phenotyping of SNTA1-deficient models across different developmental stages and stress conditions

  • Methodological solution: Integration of electrophysiological, structural, and molecular analyses in the same model systems

• Mechanistic complexities in Rac1 activation:

  • The exact sequence of events in SNTA1-mediated Rac1 activation and the relative importance of different binding partners remains unclear

  • Resolution approach: Time-resolved protein interaction studies and pathway inhibition experiments

  • Methodological solution: FRET-based biosensors to track protein interactions in real-time within living cells

• Species-specific differences:

  • Findings from mouse models don't always align with observations in human cells

  • Resolution approach: Parallel studies in both species with identical methodologies

  • Methodological solution: Humanized mouse models or advanced human tissue models (organoids)

These contradictions likely reflect the complex, context-dependent nature of SNTA1 function and highlight the need for integrated research approaches that consider tissue specificity, developmental timing, and precise molecular mechanisms.

What novel therapeutic approaches might target SNTA1 or its interaction partners in cardiac disease and cancer?

Novel therapeutic approaches targeting SNTA1 or its interaction partners show promise for both cardiac disease and cancer:

• For Long QT Syndrome (LQTS):

  • Small molecule stabilizers of the SNTA1-PMCA4b interaction could prevent the dysregulation caused by mutations like A390V

  • nNOS inhibitors specifically targeting the cardiac pool of nNOS associated with SNTA1 could reduce pathological S-nitrosylation of Nav1.5

  • Late sodium current blockers might be more effective when combined with modulators of the SNTA1 complex

  • Gene therapy approaches to deliver corrected SNTA1 to cardiomyocytes in patients with SNTA1 mutations

• For cancer treatment:

  • Tissue-specific approaches based on differential expression:

    • For breast cancer: Small molecule inhibitors of the SNTA1-P66shc-Grb2 complex or siRNA-based SNTA1 suppression

    • For esophageal cancer: SNTA1 replacement or stabilization strategies

  • Rac1 pathway modulation downstream of SNTA1 to reduce migration and invasion

  • ROS modulators targeting the SNTA1-mediated increase in reactive oxygen species

• Screening and development strategies:

  • High-throughput screens for molecules that disrupt or enhance specific SNTA1 protein interactions

  • Structure-based drug design targeting specific domains of SNTA1 involved in pathological interactions

  • Peptide mimetics that compete with specific binding interfaces

  • Targeted protein degradation approaches like PROTACs (Proteolysis-Targeting Chimeras) for context-specific SNTA1 regulation

• Delivery considerations:

  • Cardiomyocyte-targeted delivery systems for cardiac applications

  • Tumor-targeted nanoparticles for cancer applications

  • Cell-penetrating peptides for intracellular delivery of interaction-blocking molecules

Development of these therapies would benefit from the generation of high-resolution structures of SNTA1 complexes and advanced screening platforms using patient-derived cells to assess efficacy and specificity .