SNCA 1-60 Human

What exactly is SNCA 1-60 Human and how does it differ from full-length alpha-synuclein?

SNCA 1-60 Human is a deletion mutant consisting of only the first 60 amino acids of the full-length alpha-synuclein protein (which normally comprises 140 amino acids). This fragment specifically contains the N-terminal amphipathic domain of alpha-synuclein while lacking the C-terminal acidic tail (amino acids 96-140) . The truncated protein has a molecular mass of approximately 6.1 kDa, significantly smaller than the full-length protein .

The structural differences are functionally significant because recent studies have demonstrated that alpha-synuclein's chaperone activity is completely lost upon removal of its C-terminal acidic tail . This makes SNCA 1-60 particularly valuable for investigating the specific contributions of the N-terminal domain to protein function and pathology without the confounding effects of C-terminal interactions.

What are the physical and biochemical properties of purified SNCA 1-60?

SNCA 1-60 presents as a white lyophilized powder in its purified form and exhibits the following key properties:

When properly stored, SNCA 1-60 maintains a highly monomeric state, making it particularly valuable as a starting material for aggregation studies .

What are the optimal protocols for handling and storing SNCA 1-60 to maintain protein stability?

To maintain SNCA 1-60 stability during experimental work, researchers should follow these evidence-based protocols:

For short-term storage (2-4 weeks): Store at 4°C in the original buffer formulation (typically 20mM Tris-HCl pH 7.5, 100mM NaCl) .

For long-term storage: Store frozen at -20°C. To prevent degradation during multiple freeze-thaw cycles, researchers should consider:

• Adding a carrier protein (0.1% HSA or BSA) as a stabilizing agent .

• Aliquoting the protein solution into single-use volumes before freezing.

• Avoiding repeated freeze-thaw cycles, which can promote aggregation.

When thawing, samples should be brought to room temperature gradually and gently mixed rather than vortexed to avoid introducing nucleation sites for aggregation. Immediately prior to use, centrifugation at high speed (>16,000g for 10 minutes) can remove any pre-formed aggregates that might seed unwanted aggregation during experiments .

How can SNCA 1-60 be effectively used in protein aggregation studies?

SNCA 1-60 is particularly valuable for aggregation studies because it contains the N-terminal amphipathic domain while lacking the C-terminal region that normally inhibits aggregation. When designing aggregation protocols:

• Concentration effects: SNCA 1-60 should be tested at multiple concentrations (typically 20-200 μM) as aggregation kinetics are concentration-dependent.

• Buffer conditions: Standard conditions include:

  • pH: 7.4-7.5

  • Salt concentration: 100-150 mM NaCl

  • Buffer: 20 mM Tris or phosphate buffer

  • Temperature: 37°C with continuous shaking (200-300 rpm)

• Monitoring techniques:

  • Thioflavin T fluorescence for β-sheet formation

  • Dynamic light scattering for aggregate size distribution

  • Transmission electron microscopy for fibril morphology

  • Circular dichroism for secondary structure changes

• Control experiments should include:

  • Full-length alpha-synuclein for comparison

  • Buffer-only conditions to rule out artifacts

  • Known aggregation modulators as positive controls

The absence of the C-terminal domain makes SNCA 1-60 particularly useful for examining how the N-terminal region contributes to the early stages of alpha-synuclein aggregation independent of C-terminal effects .

What methods should be used to quantify SNCA 1-60 in experimental samples?

Accurate quantification of SNCA 1-60 in experimental samples can be achieved through several complementary methods:

• Western blotting:

  • Use antibodies that specifically recognize the N-terminal region of alpha-synuclein

  • The SNCA antibody raised against full-length human alpha-synuclein can recognize endogenous levels of alpha-synuclein

  • Include purified SNCA 1-60 standards at known concentrations for calibration

• ELISA:

  • Human-specific monoclonal antibodies like mAb 211 can be used for selective quantification

  • A standard curve should be generated using purified SNCA 1-60 protein

  • This method can detect concentrations as low as 0.1 ng/μl in homogenates

• Mass spectrometry:

  • Native top-down mass spectrometry can be employed for precise quantification and to assess post-translational modifications

  • Ion mobility MS can characterize metal binding and conformational states

When working with biological samples containing both endogenous full-length alpha-synuclein and SNCA 1-60, size-based separation methods should be employed prior to quantification to avoid cross-reactivity. In transgenic models, human-specific antibodies can distinguish between endogenous mouse SNCA and human SNCA 1-60 .

How does SNCA 1-60 contribute to our understanding of Parkinson's disease mechanisms?

SNCA 1-60 provides valuable insights into Parkinson's disease mechanisms through several research approaches:

• Structural basis of aggregation: The N-terminal region contained in SNCA 1-60 plays a crucial role in the initial stages of alpha-synuclein aggregation, a hallmark of Parkinson's disease. Studies with SNCA 1-60 allow researchers to isolate the contribution of this domain to pathological aggregation processes .

• Transgenic models: Researchers have created transgenic mice expressing human SNCA variants with different regulatory elements (such as the Rep1 microsatellite) to understand how gene regulation affects SNCA expression levels. These studies have shown that expanded Rep1 alleles lead to increased SNCA expression in the brain, potentially mimicking the gene multiplication seen in some familial Parkinson's cases .

• Membrane interactions: SNCA 1-60 contains the membrane-binding domain of alpha-synuclein, allowing researchers to study how membrane interactions might contribute to the protein's normal function and pathological conversion. This is particularly relevant as alpha-synuclein normally functions in regulating synaptic vesicle trafficking .

These research approaches have collectively demonstrated that the region contained within SNCA 1-60 is critical for both normal protein function and pathological processes, making it an important tool for understanding the molecular basis of Parkinson's disease.

What is the relationship between SNCA 1-60 and transcriptional regulation of the SNCA gene?

While SNCA 1-60 is a protein fragment rather than a transcriptional regulator itself, studies using transgenic models expressing this protein have revealed important insights about SNCA gene regulation relevant to Parkinson's disease:

• Rep1 microsatellite regulation: Research using transgenic mice has shown that the polymorphic Rep1 microsatellite upstream of the SNCA gene significantly influences protein expression levels. Specifically:

  • The expanded 261 bp Rep1 allele (associated with increased Parkinson's risk) resulted in 1.7-fold higher mRNA levels and 1.25-fold higher protein levels compared to the shorter 259 bp allele .

  • When accounting for total SNCA protein, the expanded risk allele contributed 2.6-fold more to SNCA steady-state levels than the shorter protective allele .

  • Targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain .

• Tissue-specific regulation: Interestingly, the Rep1 effect was not observed in blood lysates from the same mice, suggesting tissue-specific regulatory mechanisms that could explain why some SNCA mutations have neural-specific effects .

• Relevance to human disease: These findings suggest that homozygosity for the expanded Rep1 allele may functionally mimic SNCA locus multiplication, a known cause of familial Parkinson's disease, thereby explaining how this genetic variant increases PD risk .

This research demonstrates that regulatory elements controlling SNCA expression levels are critical determinants of disease risk, providing potential targets for therapeutic intervention.

How can SNCA 1-60 be used to develop seeding assays for alpha-synuclein pathology?

SNCA 1-60 can be effectively utilized in developing seeding assays that model the prion-like spread of alpha-synuclein pathology:

• Seed preparation protocol:

  • Prepare monomeric SNCA 1-60 (1 mg/ml) in aggregation buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl)

  • Incubate at 37°C with constant agitation (300 rpm) for 3-7 days

  • Sonicate the resulting fibrils to create uniform seeds

  • Verify seed formation by electron microscopy and Thioflavin T binding

• Seeding experimental design:

  • Add preformed SNCA 1-60 seeds (typically 1-10% of monomer concentration) to monomeric full-length alpha-synuclein

  • Monitor aggregation kinetics via Thioflavin T fluorescence

  • Compare seeding efficiency between SNCA 1-60-derived seeds and full-length alpha-synuclein seeds

  • Analyze the morphology of resulting aggregates by electron microscopy

• Cell-based assays:

  • Apply SNCA 1-60 seeds to neuronal cultures expressing full-length alpha-synuclein

  • Assess intracellular aggregate formation using immunocytochemistry

  • Evaluate cellular toxicity using viability assays

  • Compare pathological outcomes with those induced by patient-derived alpha-synuclein aggregates

• Translational applications:

  • Develop high-throughput screening platforms for compounds that inhibit SNCA 1-60-mediated seeding

  • Test potential therapeutic interventions that target the N-terminal domain of alpha-synuclein

  • Use in diagnostic assays for detecting pathological alpha-synuclein species in patient samples

The N-terminal region contained within SNCA 1-60 appears to be critical for the initial binding events in seeded aggregation, making this fragment particularly valuable for studying the molecular mechanisms of pathological alpha-synuclein spreading .

What techniques can be used to study the interaction between SNCA 1-60 and lipid membranes?

The N-terminal amphipathic domain contained within SNCA 1-60 is crucial for alpha-synuclein's interaction with lipid membranes. Several sophisticated techniques can be employed to study these interactions:

• Circular Dichroism (CD) Spectroscopy:

  • SNCA 1-60 undergoes a conformational change from random coil to alpha-helix upon membrane binding

  • Experimental setup should include:

    • SNCA 1-60 (5-20 μM) in buffer

    • Lipid vesicles (typically phosphatidylcholine/phosphatidylserine mixtures)

    • Measurements at 190-260 nm wavelength range

    • Controls with full-length alpha-synuclein for comparison

• Surface Plasmon Resonance (SPR):

  • Allows real-time measurement of binding kinetics

  • Lipid bilayers can be formed on L1 sensor chips

  • Association and dissociation rate constants can be determined

  • Different lipid compositions can be systematically tested

• Fluorescence Techniques:

  • Intrinsic tryptophan fluorescence (if tryptophan mutants are created)

  • Environmentally sensitive dyes like 1-anilinonaphthalene-8-sulfonic acid (ANS)

  • Förster resonance energy transfer (FRET) between labeled SNCA 1-60 and membrane components

• Atomic Force Microscopy (AFM):

  • Direct visualization of SNCA 1-60 binding to supported lipid bilayers

  • Can detect membrane remodeling events induced by protein binding

  • Allows force measurements of protein-membrane interactions

These methods collectively provide a comprehensive understanding of how the N-terminal domain in SNCA 1-60 mediates membrane interactions, which is essential for both normal function and pathological processes .

How does SNCA 1-60 differ from other alpha-synuclein fragments in terms of metal binding properties?

The metal binding properties of SNCA 1-60 differ significantly from those of full-length alpha-synuclein and other fragments:

• Metal binding sites:

  • SNCA 1-60 contains high-affinity binding sites for copper and iron predominantly in the N-terminal region

  • The fragment lacks the C-terminal binding sites for calcium and other divalent metals

  • This makes SNCA 1-60 valuable for isolating and studying N-terminal specific metal interactions

• Experimental approaches for studying metal binding:

  • Native top-down mass spectrometry has been used to characterize cobalt and manganese binding to alpha-synuclein

  • Ion mobility MS can detect conformational changes induced by metal binding

  • Isothermal titration calorimetry (ITC) can determine binding constants

  • Electron paramagnetic resonance (EPR) spectroscopy for paramagnetic metals

• Functional implications:

  • Metal binding to the N-terminal region (present in SNCA 1-60) can accelerate protein aggregation

  • This region's metal interactions may contribute to oxidative stress mechanisms in Parkinson's disease

  • Understanding these specific interactions could lead to metal-chelating therapeutic approaches

When designing experiments to study metal binding to SNCA 1-60, researchers should control for buffer composition, pH, and metal contamination in reagents, as these factors can significantly influence binding characteristics and subsequent aggregation behavior .

What are the current challenges and limitations when working with SNCA 1-60 in experimental settings?

Despite its utility, researchers face several challenges when working with SNCA 1-60:

• Aggregation control:

  • SNCA 1-60 may have different aggregation kinetics compared to full-length protein

  • Batch-to-batch variation can affect aggregation propensity

  • Unintended pre-formed aggregates may serve as seeds

  • Mitigation strategy: Always filter or centrifuge solutions immediately before use

• Physiological relevance:

  • While truncated forms of alpha-synuclein have been found in vivo, SNCA 1-60 specifically may not correspond exactly to physiologically relevant fragments

  • The lack of the C-terminal domain alters interactions with other proteins and cellular components

  • Researchers should validate findings with full-length protein in parallel experiments

• Quantification challenges:

  • Antibody cross-reactivity between SNCA 1-60 and endogenous alpha-synuclein can complicate quantification

  • The protein's intrinsic disorder makes some traditional protein assays less reliable

  • Solution: Use multiple orthogonal quantification methods and specific antibodies

• Translation to disease models:

  • Cellular uptake of SNCA 1-60 may differ from full-length protein

  • The fragment may not fully recapitulate the pathological effects seen in disease

  • Animal models expressing only SNCA 1-60 may not develop typical synucleinopathy features

These challenges necessitate careful experimental design and appropriate controls when using SNCA 1-60 as a research tool for understanding alpha-synuclein biology and pathology .

How is SNCA 1-60 being used to explore the relationship between autophagy and alpha-synuclein pathology?

Recent research has begun exploring the relationship between SNCA 1-60, autophagy, and alpha-synuclein clearance mechanisms:

• Autophagy-mediated clearance studies:

  • SNCA has been implicated in promoting autophagy-mediated cell proliferation through various signaling pathways

  • SNCA 1-60 can be used to determine whether the N-terminal domain alone is sufficient to influence autophagy

  • Experimental approaches include:

    • Monitoring LC3-II conversion in cells exposed to SNCA 1-60

    • Assessing autophagosome formation using transmission electron microscopy

    • Tracking autophagy flux using tandem fluorescent-tagged LC3

• Cellular stress models:

  • SNCA 1-60 can be employed in models of chronic inflammatory stress to study the relationship between inflammation, autophagy activation, and protein clearance

  • This approach helps determine whether autophagy upregulation is protective or detrimental in the context of alpha-synuclein accumulation

• Therapeutic implications:

  • Understanding domain-specific effects on autophagy could inform targeted therapeutic approaches

  • If the N-terminal domain (contained in SNCA 1-60) specifically modulates autophagy, drugs targeting this region might enhance protein clearance

These emerging studies suggest that specific domains of alpha-synuclein may differentially affect cellular clearance mechanisms, with important implications for therapeutic development in synucleinopathies .

What genetic approaches are being used to understand SNCA regulation that complement SNCA 1-60 protein studies?

Genetic approaches provide crucial context for SNCA 1-60 protein studies:

• SNCA-Rep1 polymorphism studies:

  • The expansion of SNCA-Rep1, an upstream polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic Parkinson's disease

  • Transgenic mouse models expressing human SNCA with different Rep1 alleles have shown:

    • mRNA levels increased 1.7-fold in homozygotes for the expanded PD risk-conferring allele compared to the shorter protective allele

    • Protein levels increased 1.25-fold in the expanded allele carriers

    • The expanded risk allele contributed 2.6-fold more to SNCA steady-state levels

• Tissue-specific regulation:

  • Interestingly, Rep1 effects were observed in brain tissue but not in blood from the same transgenic mice

  • This suggests complex tissue-specific regulatory mechanisms that could explain selective neuronal vulnerability

• Integration with SNCA 1-60 studies:

  • Combining genetic approaches with SNCA 1-60 protein studies allows researchers to understand how altered expression levels interact with protein properties

  • For example, whether increased expression of alpha-synuclein due to Rep1 expansion enhances the aggregation propensity of the N-terminal domain

• Therapeutic targeting:

  • Understanding genetic regulation mechanisms suggests potential upstream therapeutic targets to reduce SNCA expression

  • This approach may complement strategies targeting the protein itself

These genetic studies provide essential context for protein-level investigations using SNCA 1-60, highlighting the importance of integrated approaches to understanding synucleinopathies .