Recombinant Ateles geoffroyi Alpha-synuclein (SNCA)

What is the significance of studying Ateles geoffroyi alpha-synuclein compared to human alpha-synuclein?

Ateles geoffroyi (spider monkey) alpha-synuclein serves as an important comparative model in neurodegenerative research. While human alpha-synuclein is predominantly expressed in neurons, specifically in presynaptic regions, and cycles between free, partially unfolded forms and helical, membrane-bound forms , non-human primate variants like those from Ateles geoffroyi provide evolutionary context for understanding protein function and aggregation propensity.

Methodologically, researchers should approach comparative studies by:

• Performing sequence alignment analysis to identify conserved and divergent regions

• Conducting side-by-side aggregation kinetics experiments

• Evaluating structural differences using circular dichroism and other spectroscopic techniques

• Testing cross-seeding capabilities between human and spider monkey variants

The value of this approach is that differences in primary sequence can significantly impact aggregation properties, potentially providing insights into protective or pathogenic mechanisms relevant to Parkinson's disease and other synucleinopathies.

Which expression systems are optimal for producing recombinant Ateles geoffroyi alpha-synuclein?

For recombinant alpha-synuclein production, E. coli-based expression systems are generally preferred due to their cost-effectiveness and high yield. As demonstrated in human alpha-synuclein production, BL21(DE3) strains are particularly effective when coupled with high-cell-density-based expression systems . This approach can be adapted for Ateles geoffroyi alpha-synuclein.

For optimal expression, researchers should consider:

• IPTG concentration: Typically 0.5-1 mM for induction

• Growth temperature: Lowering to 25-30°C post-induction can improve folding

• Growth media: Rich media like TB (Terrific Broth) supports higher cell density

• Harvest timing: 4-6 hours post-induction for optimal yield-quality balance

The BL21(DE3) system has been validated for human alpha-synuclein production with high reproducibility through Gage R&R (reproducibility and repeatability) validation , suggesting it would be suitable for spider monkey alpha-synuclein with appropriate optimization of expression conditions.

How can researchers verify the sequence integrity of recombinant Ateles geoffroyi alpha-synuclein?

Sequence verification is crucial for ensuring research validity when working with recombinant proteins. For Ateles geoffroyi alpha-synuclein, a multi-method approach is recommended:

• DNA-level verification:

  • Sanger sequencing of the expression construct

  • Restriction enzyme mapping to confirm vector integrity

• Protein-level verification:

  • Mass spectrometry analysis (MALDI-TOF or ESI-MS) for exact mass determination

  • N-terminal sequencing for the first 10-15 amino acids

  • Tryptic digest followed by LC-MS/MS for sequence coverage analysis

• Functional verification:

  • Circular dichroism to confirm expected secondary structure characteristics

  • ThT binding assays to verify aggregation propensity

This comprehensive approach ensures that the recombinant protein maintains the expected sequence and functional properties of native Ateles geoffroyi alpha-synuclein, minimizing experimental artifacts from sequence alterations.

What are the most effective purification strategies for recombinant Ateles geoffroyi alpha-synuclein?

Purification of recombinant alpha-synuclein requires a strategic approach to ensure high purity while maintaining native protein properties. Based on advances in human alpha-synuclein purification, a non-chromatographical method has demonstrated superior reproducibility and reduced batch-to-batch variability .

A recommended purification protocol includes:

• Cell lysis via sonication or high-pressure homogenization in appropriate buffer (typically Tris-HCl pH 8.0 with protease inhibitors)

• Heat treatment (75-80°C for 10 minutes) to precipitate most E. coli proteins while alpha-synuclein remains soluble

• Ammonium sulfate fractionation (30-50% saturation)

• Isoelectric precipitation by adjusting pH to near alpha-synuclein's isoelectric point

• Dialysis against appropriate storage buffer (typically PBS or Tris-HCl)

This approach offers advantages over traditional column chromatography methods, including higher throughput, reduced cost, and improved reproducibility. SDS-PAGE analysis should show a single band at the expected molecular weight (approximately 14-19 kDa) . For Ateles geoffroyi alpha-synuclein, these methods may require optimization based on the specific protein properties.

How can researchers assess the purity and integrity of recombinant Ateles geoffroyi alpha-synuclein preparations?

Quality control is essential for ensuring experimental reproducibility. A comprehensive assessment includes:

• Purity analysis:

  • SDS-PAGE with Coomassie staining (should show >95% purity with a single band at expected molecular weight)

  • Silver staining for detection of low-abundance contaminants

  • Western blotting with anti-alpha-synuclein antibodies

• Integrity verification:

  • Mass spectrometry to confirm exact mass and detect truncations

  • N-terminal sequencing to verify intact N-terminus

  • Dynamic light scattering to assess homogeneity and detect aggregates

• Functional characterization:

  • Circular dichroism to confirm expected secondary structure

  • Thioflavin T binding assays to verify aggregation capacity

  • NMR spectroscopy for more detailed structural analysis

This multi-technique approach, similar to the orthogonal high-end analytical methods employed for human recombinant alpha-synuclein characterization , ensures that preparations meet the necessary quality standards for research applications.

What are the optimal storage conditions for maintaining the stability of recombinant Ateles geoffroyi alpha-synuclein?

Alpha-synuclein stability is crucial for experimental reproducibility. Based on established protocols for human alpha-synuclein, the following storage recommendations apply:

• Short-term storage (1-2 weeks):

  • Store at 4°C in sterile buffer (typically PBS or Tris-HCl, pH 7.4)

  • Add sodium azide (0.02%) to prevent microbial growth

• Long-term storage:

  • Lyophilize in the presence of stabilizers (e.g., sucrose or trehalose)

  • Store lyophilized protein at -20°C or -80°C

  • Alternatively, store aliquots at -80°C in buffer containing 10% glycerol

• Avoid:

  • Repeated freeze-thaw cycles (prepare single-use aliquots)

  • Prolonged storage at room temperature

  • Exposure to extreme pH conditions

Before each experiment, researchers should verify protein integrity using techniques such as SDS-PAGE, dynamic light scattering, or CD spectroscopy to ensure that storage has not affected protein quality.

How should researchers design aggregation experiments with recombinant Ateles geoffroyi alpha-synuclein?

Aggregation studies are fundamental in alpha-synuclein research. For robust experimental design:

• Preparation conditions:

  • Use freshly purified or properly stored protein

  • Filter solutions (0.22 μm) immediately before experiments to remove preformed aggregates

  • Standardize protein concentration (typically 0.5-2.5 μM for initial experiments)

• Aggregation conditions:

  • Buffer composition (PBS or Tris-HCl pH 7.4 with controlled ionic strength)

  • Temperature (37°C is standard, but comparative studies at different temperatures provide insights into kinetics)

  • Agitation (constant orbital shaking at 200-400 rpm)

  • Presence/absence of cofactors that influence aggregation (metals, lipids, etc.)

• Monitoring techniques:

  • Thioflavin T fluorescence for real-time monitoring

  • Dynamic light scattering for particle size distribution

  • Atomic force microscopy or transmission electron microscopy for fibril morphology

  • Circular dichroism for secondary structure transitions

• Controls:

  • Positive control (pre-formed fibrils)

  • Negative control (non-aggregating protein variant)

  • Parallel experiments with human alpha-synuclein for comparison

This methodological framework enables the characterization of species-specific aggregation properties of Ateles geoffroyi alpha-synuclein compared to human variants.

What methods are most effective for detecting alpha-synuclein seeds in biological samples?

Detection of alpha-synuclein seeds in biological samples requires sensitive and specific techniques. The immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC) assay has emerged as a powerful method :

• Sample preparation:

  • For serum/plasma: immunoprecipitation with anti-alpha-synuclein antibodies

  • For CSF: direct use or concentration through ultrafiltration

  • For tissue: homogenization in appropriate buffer followed by clarification

• RT-QuIC assay setup:

  • Substrate preparation: recombinant alpha-synuclein (0.5-2.5 μM)

  • Reaction buffer: typically containing ThT for real-time monitoring

  • Plate reader setup: continuous shaking with intermittent reading

  • Controls: known positive (synucleinopathy samples) and negative samples

• Data analysis:

  • Kinetic parameters: lag time, maximum fluorescence, slope

  • Threshold determination for positive/negative classification

  • Statistical analysis accounting for technical replicates

This approach has demonstrated high diagnostic performance in differentiating Parkinson's disease and multiple system atrophy from controls, with area under the curve values of 0.93-0.96 . When adapting this method for Ateles geoffroyi alpha-synuclein, researchers should optimize antibody selection and reaction conditions accordingly.

How can researchers differentiate between different conformational strains of Ateles geoffroyi alpha-synuclein?

Strain differentiation is critical for understanding pathological mechanisms. A comprehensive approach includes:

• Structural characterization:

  • Transmission electron microscopy for fibril morphology

  • X-ray diffraction for cross-β structure details

  • Solid-state NMR for atomic-level structural differences

  • Limited proteolysis to identify protected core regions

• Functional differentiation:

  • Seeding capacity in cell-based assays

  • Cross-seeding efficiency between strains

  • Toxicity profiles in neuronal cultures

  • Binding affinity to specific antibodies or ligands

• Biophysical properties:

  • Stability in different denaturants (urea, guanidine-HCl)

  • Temperature sensitivity

  • Resistance to proteolytic degradation

This methodological framework enables detailed characterization of conformational variants, similar to approaches that have successfully differentiated alpha-synuclein conformers associated with different synucleinopathies . The analysis revealed that amplified seeds from Parkinson's disease and multiple system atrophy exhibit distinct structural and propagation properties, suggesting that disease-specific phenotypes may result from these structural differences .

How can Ateles geoffroyi alpha-synuclein be used in comparative studies to understand species-specific aspects of neurodegeneration?

Comparative studies offer unique insights into evolutionary aspects of protein function and disease susceptibility. A methodological approach includes:

• Sequence-structure-function analysis:

  • Identify key sequence differences between species

  • Model structural implications using computational approaches

  • Test functional consequences experimentally

• Cross-species seeding experiments:

  • Assess if Ateles geoffroyi alpha-synuclein can seed human alpha-synuclein aggregation and vice versa

  • Quantify seeding efficiency using ThT kinetics and lag time analysis

  • Characterize resulting fibril structures for hybrid characteristics

• Species-specific interaction studies:

  • Compare binding profiles with chaperones, lipids, and other interaction partners

  • Assess differences in post-translational modification patterns

  • Evaluate sensitivity to various stress conditions

• In vitro and cellular toxicity comparisons:

  • Standardized cytotoxicity assays using identical concentrations of each species variant

  • Assessment of membrane interaction properties

  • Evaluation of subcellular localization patterns

These approaches can reveal whether specific sequence differences confer resistance or susceptibility to aggregation, potentially identifying protective mechanisms that could inform therapeutic strategies for human synucleinopathies.

What are the key considerations for developing animal models using Ateles geoffroyi alpha-synuclein?

Animal models are essential for studying in vivo effects of alpha-synuclein variants. Key methodological considerations include:

• Expression system selection:

  • Viral vectors (AAV, lentivirus) for localized expression

  • Transgenic approaches for germline expression

  • Conditional expression systems for temporal control

• Delivery method optimization:

  • Stereotactic injection parameters (coordinates, volume, flow rate)

  • Promoter selection for cell-type specificity

  • Dosage titration to achieve physiologically relevant expression levels

• Phenotypic characterization:

  • Behavioral assessment (motor function, cognition)

  • Histopathological analysis (alpha-synuclein aggregation, neurodegeneration)

  • Biochemical analysis (soluble vs. insoluble fractions, post-translational modifications)

  • Neurophysiological evaluation (electrophysiology, calcium imaging)

• Experimental controls:

  • Empty vector controls

  • Human alpha-synuclein parallel models

  • Wild-type vs. mutant variants

This framework enables rigorous evaluation of species-specific effects of Ateles geoffroyi alpha-synuclein in vivo, potentially revealing insights into differential susceptibility to synucleinopathies across primates.

How can researchers effectively use recombinant Ateles geoffroyi alpha-synuclein in the development and validation of therapeutic strategies?

Therapeutic development requires carefully designed experimental approaches. A methodological framework includes:

• Target validation studies:

  • Identification of species-specific binding sites for small molecules

  • Comparative screening against human and Ateles geoffroyi variants

  • Structure-activity relationship studies incorporating species differences

• Aggregation inhibitor screening:

  • High-throughput ThT-based aggregation assays

  • Dose-response analysis with standardized metrics (IC50, EC50)

  • Secondary validation using orthogonal techniques (EM, DLS)

  • Counter-screening for non-specific effects

• Disaggregation agent evaluation:

  • Preformed fibril disassembly assays

  • Kinetic analysis of disassembly

  • Toxicity assessment of resulting species

• Immunotherapy approach development:

  • Epitope mapping across species variants

  • Cross-reactivity testing of antibodies

  • Fc-dependent and independent mechanisms of action

This comprehensive approach leverages the comparative aspects of Ateles geoffroyi alpha-synuclein to develop more robust therapeutic strategies with potential cross-species applicability, enhancing translational potential.

How can researchers address batch-to-batch variability in recombinant Ateles geoffroyi alpha-synuclein preparations?

Batch-to-batch variability presents a significant challenge for experimental reproducibility. A systematic approach to minimizing this variability includes:

• Standardized production protocol:

  • Implement Gage R&R validated protocols similar to those developed for human alpha-synuclein

  • Use high-cell-density-based expression systems with consistent induction parameters

  • Employ non-chromatographical purification methods demonstrated to reduce variability

• Quality control metrics:

  • Develop standard acceptance criteria for each batch

  • Implement orthogonal high-end analytical methods for characterization

  • Use principal component analysis (PCA) to assess batch similarity

• Reference standard implementation:

  • Maintain a well-characterized reference batch

  • Compare each new batch against this reference using standardized assays

  • Document relative performance in key functional tests

• Documentation and reporting:

  • Maintain detailed production records

  • Report batch information in publications

  • Share standardized protocols with the research community

This approach has successfully reduced batch-to-batch variability in human alpha-synuclein preparations and can be adapted for Ateles geoffroyi alpha-synuclein to enhance research reproducibility across laboratories.

What strategies can address the challenges of maintaining native conformational states of recombinant Ateles geoffroyi alpha-synuclein?

Alpha-synuclein is known to cycle between different conformational states, presenting challenges for maintaining native states in recombinant preparations. Methodological approaches include:

• Buffer optimization:

  • Screen various buffer compositions to identify stabilizing conditions

  • Consider the addition of osmolytes like glycerol or trehalose

  • Maintain consistent pH and ionic strength

• Storage protocol development:

  • Test different storage temperatures (-80°C, -20°C, 4°C)

  • Evaluate flash-freezing vs. slow cooling

  • Compare storage in solution vs. lyophilized state

• Pre-experimental quality checks:

  • Circular dichroism to verify secondary structure

  • Size exclusion chromatography to assess oligomeric state

  • Dynamic light scattering to detect aggregation

• Handling practices:

  • Minimize mechanical stress during handling

  • Use protein-compatible surfaces for containers

  • Establish maximum acceptable freeze-thaw cycles

These approaches ensure that experimental outcomes reflect the properties of the native protein rather than artifacts introduced during production and storage, enhancing the reliability of comparative studies between human and non-human primate alpha-synuclein variants.

How can researchers effectively distinguish between physiological and pathological forms of Ateles geoffroyi alpha-synuclein?

Distinguishing between normal and disease-associated forms is crucial for research validity. A methodological framework includes:

• Conformational analysis:

  • Circular dichroism to assess secondary structure content

  • Intrinsic fluorescence for tertiary structure evaluation

  • EPR spectroscopy with spin labeling for detailed conformational analysis

• Aggregation state characterization:

  • Size exclusion chromatography for oligomeric state distribution

  • Analytical ultracentrifugation for precise molecular weight determination

  • Native PAGE for visualization of different assemblies

• Functional assays:

  • Membrane binding studies (lipid vesicle association)

  • Synaptic vesicle recycling assays

  • Interaction analysis with physiological binding partners

• Pathological markers:

  • Thioflavin T/S binding

  • Congo red birefringence

  • Seeding capacity in RT-QuIC assays

  • Proteinase K resistance profiles

This integrated approach enables researchers to characterize the continuum between physiological and pathological forms, similar to methods that have successfully differentiated normal from disease-associated human alpha-synuclein . The identification of specific markers of pathological forms supports the development of biological definitions of alpha-synuclein-related diseases .

How can advanced structural biology techniques be applied to study species-specific differences in alpha-synuclein?

Cutting-edge structural biology approaches offer unprecedented insights into alpha-synuclein biology. A methodological framework includes:

• Cryo-EM analysis:

  • Sample preparation optimization for fibrillar assemblies

  • Data collection parameters for optimal resolution

  • Image processing workflows for heterogeneous samples

  • Structural comparison between species variants

• NMR spectroscopy:

  • Sample isotope labeling strategies

  • Resonance assignment approaches

  • Dynamics measurements on multiple timescales

  • Mapping of species-specific differences in dynamics and structure

• Hydrogen-deuterium exchange mass spectrometry:

  • Optimization of exchange conditions

  • Quench and digestion protocols

  • Data analysis for conformational flexibility comparison

  • Integration with computational modeling

• In-cell structural studies:

  • Development of cellular expression systems

  • In-cell NMR approaches

  • Proximity labeling methods

  • Live-cell imaging of conformational sensors

These advanced methods enable detailed comparison of structural features between human and Ateles geoffroyi alpha-synuclein variants, potentially revealing species-specific determinants of aggregation propensity and pathogenicity.

What are the methodological approaches for investigating the role of post-translational modifications in Ateles geoffroyi alpha-synuclein function?

Post-translational modifications (PTMs) critically influence alpha-synuclein behavior. A comprehensive methodology includes:

• PTM mapping:

  • Mass spectrometry-based proteomics for PTM identification

  • Site-specific antibodies for PTM detection

  • Comparison of PTM patterns across species

• Generation of modified recombinant proteins:

  • Enzymatic modification in vitro

  • Genetic code expansion for site-specific incorporation

  • Chemical conjugation approaches

  • Semisynthetic protein ligation methods

• Functional impact assessment:

  • Aggregation kinetics of modified variants

  • Membrane binding properties

  • Interaction with cellular partners

  • Subcellular localization patterns

• In vivo relevance:

  • Development of PTM-specific detection methods for biological samples

  • Correlation of specific PTMs with disease states

  • Temporal dynamics of modifications during disease progression

This framework enables detailed investigation of how species-specific differences in PTM patterns might contribute to differential susceptibility to synucleinopathies, potentially identifying protective modifications in non-human primate alpha-synuclein variants.

How can systems biology approaches integrate Ateles geoffroyi alpha-synuclein data into broader understanding of synucleinopathies?

Systems biology offers powerful tools for contextualizing species-specific findings. Methodological approaches include:

• Interactome analysis:

  • Affinity purification-mass spectrometry to identify binding partners

  • Yeast two-hybrid screening for interaction mapping

  • Comparison of interactomes between species

  • Network analysis to identify conserved and divergent pathways

• Multi-omics integration:

  • Transcriptomic responses to alpha-synuclein expression

  • Proteomic changes in cellular models

  • Metabolomic alterations associated with alpha-synuclein pathology

  • Integration of datasets using computational approaches

• Mathematical modeling:

  • Kinetic models of aggregation pathways

  • Agent-based modeling of cellular responses

  • Population-level models of disease progression

  • Comparative modeling across species

• Translational applications:

  • Identification of biomarker candidates

  • Drug target prioritization

  • Prediction of therapeutic response

  • Patient stratification strategies

This integrative approach contextualizes species-specific findings within broader biological systems, enhancing translational relevance and providing a framework for understanding how evolutionary differences in alpha-synuclein contribute to disease susceptibility and progression.