Recombinant Pongo abelii Syntaxin-1A (STX1A), partial

What is the molecular function of Syntaxin-1A in neuronal systems?

Syntaxin-1A functions as a key component of the SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex, which mediates synaptic vesicle fusion and neurotransmitter release. It contains both a SNARE domain and an Habc domain. The Habc domain interacts with syntaxin binding protein 1 (STXBP1, also known as MUNC18-1) to regulate SNARE complex formation, vesicle docking, and release . Syntaxin-1A plays a crucial role in the presynaptic vesicle fusion apparatus, which is essential for the calcium-dependent exocytosis of neurotransmitters and neuropeptides . In nociceptive neurons, STX1A is involved in the release of neuropeptides such as Calcitonin Gene-Related Peptide (CGRP) in response to stimuli like capsaicin activation of TRPV1 channels .

How does recombinant Pongo abelii STX1A compare structurally to human STX1A?

While specific comparison data between Pongo abelii (Sumatran orangutan) STX1A and human STX1A is not explicitly detailed in the literature, recombinant protein research typically focuses on conserved domains and functional regions. Human STX1A has a well-characterized amino acid sequence including the expressed region: MKDRTQELRTAKDSDDDDDVAVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEEVKRKHSAILASPNPDEKTKEELEELMSDI KKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGRTTTSEELE DMLESGNPAIFASGIIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQ . Researchers working with Pongo abelii STX1A should note that while primate STX1A proteins share high homology, species-specific variations may affect protein-protein interactions and functional characteristics, necessitating careful validation when extrapolating findings between species.

What expression systems are most suitable for producing recombinant Pongo abelii STX1A?

For recombinant STX1A expression, E. coli systems have been successfully employed for human STX1A production, as indicated by commercial availability of such preparations . Methodology for Pongo abelii STX1A would follow similar protocols with species-specific considerations:

The optimal expression system selection should be guided by the specific research requirements, considering factors such as required protein yield, need for post-translational modifications, and intended downstream applications .

What purification strategies yield the highest purity of functional recombinant STX1A?

For high-purity recombinant STX1A preparation, a multi-step purification process is recommended:

• Initial Capture: Affinity chromatography using His-tag or GST-tag fusion constructs

• Intermediate Purification: Ion exchange chromatography to separate based on charge differences

• Polishing Step: Size exclusion chromatography to achieve final purity

Quality assessment should include:

• RP-HPLC analysis to confirm >95% purity

• SDS-PAGE verification

• Western blot confirmation of identity

• Functional assays to verify biological activity

A typical formulation for purified STX1A includes 20mM Tris-HCl pH7.5, 10% glycerol, and 1mM DTT to maintain protein stability . Researchers should verify protein activity through binding assays with known STX1A interaction partners such as SNAP-25 and synaptobrevin to ensure functional integrity of the purified protein.

How can researchers effectively use recombinant Pongo abelii STX1A to study evolutionary conservation of synaptic machinery?

Comparative analysis of Pongo abelii STX1A with human and other primate STX1A proteins can provide valuable insights into the evolution of the synaptic machinery. Methodological approach:

• Sequence Analysis:

  • Perform phylogenetic analysis of STX1A across primate species

  • Identify conserved domains vs. variable regions

  • Map conservation patterns to functional domains

• Functional Conservation Assessment:

  • Test cross-species interactions with binding partners

  • Design chimeric proteins swapping domains between species

  • Utilize in vitro vesicle fusion assays to compare functional capacities

• Structural Biology Approach:

  • Compare X-ray crystallography or cryo-EM structures

  • Examine differences in protein folding and domain organization

  • Correlate structural variations with functional differences

These approaches can reveal whether variations in STX1A across primates contribute to species-specific neural processing capabilities and provide insights into human-specific neurological conditions .

What experimental approaches can determine the role of Pongo abelii STX1A in pain signaling pathways?

Based on findings that STX1A is implicated in pain sensitivity in humans, investigators can design experiments to study its role in Pongo abelii pain signaling:

• Primary Neuron Cultures:

  • Establish primary sensory neuron cultures from Pongo abelii tissue or use human neurons with recombinant Pongo abelii STX1A

  • Utilize lentiviral vectors to systematically vary STX1A expression levels

  • Measure capsaicin-evoked CGRP release as a functional readout of nociceptive function

• Electrophysiological Studies:

  • Conduct patch-clamp recordings to assess synaptic transmission in the presence of varying concentrations of recombinant STX1A

  • Analyze changes in calcium signaling dynamics using calcium imaging techniques

• Molecular Interaction Analysis:

  • Perform co-immunoprecipitation studies to identify species-specific interaction partners

  • Use proximity ligation assays to visualize protein interactions in situ

  • Compare STX1A interactions with TRPV1 and other pain signaling molecules

The biphasic effect observed in human studies—where low supplementation enhanced release while higher levels inhibited it—suggests a dose-dependent mechanism that should be carefully examined in cross-species comparisons .

How does SNP variation in STX1A affect protein function, and what methodologies can assess these effects in recombinant systems?

Studies have identified STX1A SNPs as risk factors for conditions like migraine. To investigate functional consequences of these variants:

• Site-Directed Mutagenesis Strategy:

  • Create recombinant Pongo abelii STX1A proteins carrying equivalent SNPs to human rs941298 and rs6951030

  • Express wild-type and variant proteins in the same system

  • Compare protein stability, expression levels, and subcellular localization

• Functional Assays:

  • Measure vesicle fusion efficiency using fluorescence-based assays

  • Assess interaction strength with binding partners using surface plasmon resonance

  • Conduct calcium imaging to evaluate effects on calcium-dependent exocytosis

• Structural Analysis:

  • Perform circular dichroism spectroscopy to detect changes in protein secondary structure

  • Use nuclear magnetic resonance to identify conformational changes

  • Conduct molecular dynamics simulations to predict functional consequences

This systematic approach can reveal how specific SNPs may alter STX1A function across species and potentially contribute to neurological disorder susceptibility .

What mechanisms explain the inhibition of vesicle fusion by STX1A overexpression, and how can these be studied using recombinant proteins?

STX1A overexpression has been shown to inhibit dense-core vesicle release, potentially through a "dominant-negative synaptopathy" or SNAREopathy mechanism . To investigate this using recombinant Pongo abelii STX1A:

• In Vitro Reconstitution Systems:

  • Develop liposome-based fusion assays with purified recombinant proteins

  • Systematically vary STX1A concentration while keeping other SNARE proteins constant

  • Measure fusion kinetics using fluorescence dequenching assays

• Domain-Specific Analysis:

  • Create truncated constructs expressing only the Habc domain

  • Test competitive interference with full-length STX1A function

  • Examine effects on clustering of vesicle release machinery at plasma membrane

• Interaction with Calcium Channels:

  • Investigate binding to the synaptic protein interaction (synprint) domain of voltage-gated Ca²⁺ channels

  • Assess spatial coupling between calcium influx and vesicle docking

  • Evaluate effects on the temporal dynamics of the release process

These approaches will help delineate the mechanisms by which STX1A overexpression disrupts the presynaptic vesicle fusion apparatus, with potential implications for understanding neurological conditions characterized by altered neurotransmitter release .

How can recombinant Pongo abelii STX1A be used in screening assays for novel pain therapeutics?

Given STX1A's role in pain sensitivity, recombinant protein can be utilized in drug discovery platforms:

• High-Throughput Binding Assays:

  • Immobilize recombinant STX1A on biosensor chips

  • Screen compound libraries for molecules that modulate STX1A interactions

  • Develop fluorescence polarization assays to detect binding of small molecules

• Functional Screening Systems:

  • Establish cell-based assays expressing recombinant STX1A and nociceptive markers

  • Measure CGRP release in response to capsaicin with and without test compounds

  • Develop calcium flux assays in STX1A-expressing sensory neurons

• Target Validation Approach:

  • Compare effects across species (human vs. Pongo abelii STX1A)

  • Test compounds in both gain-of-function and loss-of-function STX1A models

  • Validate hits in more complex systems (tissue explants, animal models)

The dose-response relationships observed in primary cultures suggest that fine-tuning STX1A activity, rather than complete inhibition, may be the optimal therapeutic strategy for pain modulation .

What are the critical quality control parameters for recombinant Pongo abelii STX1A preparation?

Ensuring consistent quality of recombinant STX1A requires rigorous quality control procedures:

Common challenges include protein aggregation during concentration steps and loss of activity during freeze-thaw cycles. To preserve activity, store recombinant STX1A in small aliquots at -80°C in buffer containing 20mM Tris-HCl pH7.5, 10% glycerol, and 1mM DTT . Avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of functional activity.

How can researchers optimize transfection of STX1A constructs for co-expression with TRPV1 in nociceptive neurons?

For studying STX1A-TRPV1 interactions in pain signaling pathways, optimized co-expression is essential:

• Vector Design Considerations:

  • Use bicistronic constructs with IRES or 2A peptide sequences

  • Consider dual promoter systems for independent expression control

  • Include fluorescent tags to confirm co-expression (e.g., STX1A-GFP, TRPV1-mCherry)

• Transfection Protocol Optimization:

  • For primary DRG neurons: utilize nucleofection or magnetofection for higher efficiency

  • Adjust DNA ratios to achieve physiologically relevant expression levels

  • Implement staggered transfection if protein expression kinetics differ

• Validation Methods:

  • Perform multiplex fluorescent in situ hybridization to verify co-expression

  • Use quantitative immunocytochemistry to assess protein levels

  • Conduct single-cell RT-PCR to confirm transcript presence

Based on human DRG neuron studies showing complete overlap of STX1A expression with TRPV1, researchers should aim for similar co-expression patterns when establishing model systems to ensure physiological relevance .

How might comparative studies of STX1A across primates inform human neurological disorder research?

Cross-species analysis of STX1A function offers valuable insights into both evolutionary neurobiology and human pathology:

• Neurological Disorder Connections:

  • Investigate whether species variations in STX1A correlate with differences in pain sensitivity

  • Explore potential links between STX1A and migraine susceptibility across primates

  • Examine STX1A variants in the context of 7q11.23 duplication syndrome equivalent regions in non-human primates

• Methodological Approach:

  • Conduct systematic phylogenetic analysis of STX1A across primate species

  • Develop equivalent experimental paradigms for cross-species functional comparisons

  • Utilize CRISPR-based approaches to humanize STX1A in model organisms

• Translational Implications:

  • Identify conserved vs. divergent regulatory mechanisms

  • Develop more precise animal models for STX1A-related disorders

  • Discover novel therapeutic targets based on species-specific adaptations

The connection between STX1A duplications and pain insensitivity in humans provides a compelling rationale for investigating whether similar genotype-phenotype correlations exist in other primates, potentially revealing evolutionary adaptations in pain processing mechanisms .

What are the emerging technologies that could advance recombinant STX1A research?

Several cutting-edge approaches show promise for advancing STX1A research:

• Cryo-Electron Microscopy:

  • Visualize the complete SNARE complex architecture at near-atomic resolution

  • Capture different conformational states during the fusion process

  • Resolve species-specific structural variations

• Optogenetic Tools:

  • Develop light-sensitive STX1A variants for temporal control of function

  • Create optically controlled dimerization systems to manipulate SNARE complex formation

  • Enable precise spatiotemporal control of synaptic release machinery

• Organoid Models:

  • Generate species-specific brain organoids expressing native or modified STX1A

  • Create pain pathway organoids to study nociceptive signaling

  • Develop multi-cellular systems to examine STX1A function in complex neural circuits

• Single-Molecule Imaging Techniques:

  • Track individual STX1A molecules during vesicle docking and fusion

  • Measure protein-protein interaction kinetics in living cells

  • Visualize nanoscale organization of release sites

These advanced techniques will facilitate deeper understanding of STX1A function in health and disease, potentially leading to novel therapeutic strategies for pain management and neurological disorders .