SNAPIN Human
Description
Functional Roles of SNAPIN
Key Pathways and Interactions
Pancreatic β-Cell Proliferation
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Overexpression: Increases β-cell proliferation by 2.5-fold, elevates S-phase cells from 6.85% to 27.13%, and enhances Cyclin D1/CDK2 expression .
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Knockdown: Reduces insulin secretion by 40–50% and increases apoptosis by 50% .
Lysosomal Dysregulation in Macrophages
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SNAPIN silencing: Causes lysosomal alkalinization (pH 6.2 → 7.1), impaired cathepsin D activation, and autophagosome accumulation .
Neurological Implications
Product Specs
Q&A
What is the molecular structure of SNAPIN and how should researchers approach its characterization?
SNAPIN is a relatively small protein with a molecular weight of approximately 15 kDa, consisting of 136 amino acids . It is enriched in neurons and exclusively located on synaptic vesicle membranes .
Methodological approach: Researchers should employ a combination of techniques to characterize SNAPIN:
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X-ray crystallography or NMR spectroscopy for structural determination
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Immunoblotting with specific anti-SNAPIN antibodies for detection
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Subcellular fractionation to confirm synaptic vesicle localization
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Density gradient centrifugation for isolation of SNAPIN-containing compartments
How does SNAPIN interact with the SNARE complex and what experimental designs best demonstrate this interaction?
SNAPIN was originally discovered as a receptor protein for SNAP-25 in the SNARE complex . By binding to SNAP-25, SNAPIN facilitates stable binding of the SNARE complex to synaptotagmin-1, promoting vesicle docking .
Recommended experimental design:
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Co-immunoprecipitation assays with SNAP-25 antibodies
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Proximity ligation assays to visualize interactions in situ
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In vitro binding assays with purified components
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FRET or BRET analysis to measure direct protein interactions
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Site-directed mutagenesis to identify critical binding domains
| Technique | Application | Advantages | Limitations |
|---|---|---|---|
| Co-IP | Physical interaction | Detects endogenous complexes | May capture indirect interactions |
| Proximity Ligation | In situ visualization | Maintains cellular context | Requires optimization |
| FRET/BRET | Direct interaction | Real-time dynamics | Technical complexity |
| Mutagenesis | Binding domain mapping | Precise mechanism | Potential structural disruption |
What experimental approaches can effectively demonstrate SNAPIN's role in synaptic development?
SNAPIN plays a crucial role in the normal growth and development of synapses . Researchers should design experiments that address both structural and functional aspects of synaptic development.
Methodological approach:
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Primary neuronal cultures with SNAPIN knockdown or overexpression
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Time-lapse imaging of synapse formation in developing neurons
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Electrophysiological recordings to assess functional maturation
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Electron microscopy to evaluate ultrastructural changes
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Immunostaining for pre/postsynaptic markers to quantify synapse density
How can researchers resolve contradictory findings regarding SNAPIN's role in neurotransmitter release?
The search results indicate that the necessity of SNAPIN for proper synaptic release varies across species , which may explain conflicting data in the literature.
Recommended approach to resolve contradictions:
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Comparative studies across multiple model organisms
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Standardized experimental conditions (temperature, calcium concentration)
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Rescue experiments with species-specific SNAPIN variants
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Careful quantification of release parameters (probability, kinetics)
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Consideration of compensatory mechanisms in different genetic backgrounds
| Species | SNAPIN Dependency | Experimental System | Key Findings |
|---|---|---|---|
| Mouse | High | Hippocampal cultures | Essential for vesicle priming |
| Rat | Moderate | Cortical neurons | Important for release probability |
| Human | Variable | iPSC-derived neurons | Context-dependent function |
What methods reveal SNAPIN's role in autophagosome maturation and how should experimental design address this function?
SNAPIN is critical for autophagosome maturation. When SNAPIN is reduced, late autophagosome vacuoles containing partially digested organelles accumulate .
Optimal experimental design:
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Electron microscopy to directly visualize autophagosome accumulation
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LC3-II/LC3-I ratio quantification by Western blot
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Immunofluorescence to measure LC3 puncta formation
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Density gradient centrifugation for lysosome isolation
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Purification of autophagosomes using anti-LC3B antibody and magnetic beads
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Comparative analysis between control and SNAPIN-deficient cells
How does SNAPIN contribute to brain homeostasis and what experimental approaches best demonstrate this function?
SNAPIN helps maintain brain homeostasis in synaptic transmission, neural development, neural protection, and learning and memory .
Methodological approach:
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Conditional SNAPIN knockout in specific brain regions
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Behavioral testing for learning and memory performance
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In vivo calcium imaging to assess neuronal activity homeostasis
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Transcriptomic analysis of homeostatic gene expression
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Assessment of stress responses in SNAPIN-deficient neurons
What methodological approaches best demonstrate SNAPIN's involvement in Alzheimer's disease pathology?
Snapin-deficient mouse brains recapitulate Alzheimer's disease (AD)-associated autophagic stress in axons, and overexpressing SNAPIN reversed this stress .
Recommended experimental design:
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Comparative analysis of SNAPIN expression in AD versus control brain tissue
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Assessment of autophagy markers in SNAPIN-deficient models
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Live imaging of autophagosome transport in AD model neurons
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SNAPIN overexpression in AD models to evaluate therapeutic potential
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Correlation of SNAPIN levels with cognitive measures in AD models
How can researchers effectively investigate SNAPIN's role in synaptic homeostasis related to schizophrenia?
SNAPIN expression levels are associated with vesicle priming and synaptic homeostasis under high-frequency stimulation, potentially causing cognitive impairment in schizophrenia .
Methodological approach:
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Analysis of SNAPIN expression in postmortem schizophrenia brain samples
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Electrophysiological recordings under high-frequency stimulation
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Manipulation of SNAPIN levels in neuronal cultures from patient-derived iPSCs
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Assessment of cognitive domains in animal models with altered SNAPIN expression
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Correlation of SNAPIN polymorphisms with schizophrenia endophenotypes
How can researchers effectively study SNAPIN's interaction with dynein motors in BDNF retrograde signaling?
SNAPIN, as a dynein adapter, recruits dynein motors to BDNF-TrkB signaling endosomes, assisting with terminal BDNF-induced retrograde signaling crucial for dendritic growth of cortical neurons .
Recommended experimental design:
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Microfluidic chambers to physically separate axons from cell bodies
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Live imaging of fluorescently tagged SNAPIN and dynein components
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Compartmentalized BDNF application to axon terminals
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Analysis of retrograde signaling using phospho-CREB immunostaining
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Co-immunoprecipitation of SNAPIN with dynein and TrkB receptors
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Quantification of dendritic arborization following manipulation of SNAPIN-dynein interaction
What experimental design considerations are important when studying SNAPIN's role in cellular differentiation?
Research indicates SNAPIN is required for functional autophagy and is critical for monocyte to macrophage differentiation .
Methodological approach:
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Isolation of peripheral blood monocytes by counter-flow elutriation
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Differentiation induction with 20% FBS plus CSF-1
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SNAPIN knockdown using siRNA transfection at specific timepoints
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Assessment of differentiation markers (CD163, CD71) by flow cytometry
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Morphological evaluation of differentiation progression
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Comparison with other autophagy regulators (e.g., Beclin 1)
| Differentiation Stage | Key Markers | SNAPIN Requirement | Method of Assessment |
|---|---|---|---|
| Early monocyte | CD14+/CD16- | Low | Flow cytometry |
| Intermediate | CD14+/CD71+ | Moderate | Immunofluorescence |
| Mature macrophage | CD163+/CD71+ | High | Flow cytometry, morphology |
How can researchers design experiments to study the regulation of SNAPIN by protein kinase A?
SNAPIN is a substrate for protein kinase A (PKA), which can regulate neurotransmitter release by directly acting on relevant proteins .
Recommended experimental design:
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In vitro phosphorylation assays with purified SNAPIN and PKA
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Site-directed mutagenesis of potential phosphorylation sites
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Pharmacological manipulation of PKA activity in neuronal cultures
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Analysis of SNAPIN phosphorylation state using phospho-specific antibodies
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Functional assessment of neurotransmitter release with phosphomimetic SNAPIN variants
Product Science Overview
Synaptosomal-Associated Protein 25 (SNAP-25) is a crucial protein involved in the regulation of neurotransmitter release at synapses. It is encoded by the SNAP25 gene located on chromosome 20p12.2 in humans . This protein is a member of the SNAP-25 family and plays a significant role in the SNARE (Soluble NSF Attachment Protein Receptor) complex, which is essential for synaptic vesicle fusion and neurotransmitter release .
SNAP-25 is a membrane-bound protein anchored to the cytosolic face of membranes via palmitoyl side chains in the middle of the molecule . It contains two t-SNARE coiled-coil homology domains, which are critical for its function in the SNARE complex . The SNARE complex is responsible for the specificity of membrane fusion and directly executes fusion by forming a tight complex that brings the synaptic vesicle and plasma membranes together .
Recombinant human SNAP-25 is produced using Escherichia coli (E. coli) as the host organism . The recombinant protein consists of 217 amino acids and has a calculated molecular mass of 24.8 kDa, although it migrates as an approximately 28 kDa band in SDS-PAGE under reducing conditions . The protein is typically lyophilized from sterile PBS, pH 7.4, and may contain protectants such as trehalose, mannitol, and Tween80 . It is stable for up to twelve months when stored at -20°C to -80°C under sterile conditions .
SNAP-25 is a key component of the SNARE complex, which includes syntaxin-1 and synaptobrevin . This complex is essential for the exocytotic fusion of synaptic vesicles with the presynaptic membrane, a process critical for neurotransmitter release . SNAP-25 has been implicated in various neurological disorders, including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia, bipolar disorder, and epilepsy . Its role in these conditions highlights its importance as a shared biological substrate among different "synaptopathies" .
Mutations or dysregulation of the SNAP25 gene can lead to developmental and epileptic encephalopathies (DEEs), learning disabilities, and other neurological conditions . The protein’s involvement in synaptic vesicle docking and neurotransmitter release makes it a potential target for therapeutic interventions in these disorders .
