NCS1 213 a.a. Human

What is the basic structure of human NCS-1 protein?

Human NCS-1 is a 213 amino acid protein containing two pairs of EF-hand motifs, of which only three are functional: EF-2, EF-3, and EF-4. EF-2 and EF-3 can recognize both Ca2+ and Mg2+, functioning as structural sites that allow the protein to adopt its tertiary fold. EF-4 serves as a regulatory Ca2+ binding site that senses changes in cytosolic calcium levels in neurons .

The protein contains a surface-exposed hydrophobic crevice that recognizes target proteins, which typically present short helical motifs that bind to either the N- or C-terminal part of this cavity. This crevice is critical for NCS-1's ability to bind various protein partners .

How does NCS-1 differ from other neuronal calcium sensors?

Unlike other neuronal calcium sensors, NCS-1 is found both inside and outside the nervous system. It does not contain a Ca2+/Myr switch, which means it remains constantly bound to cell membranes. This characteristic enables NCS-1 to interact with multiple binding partners and participate in diverse cellular functions .

NCS-1 uses its hydrophobic crevice to recognize targets, with the shape and size of this crevice determining target specificity. The protein contains a dynamic C-terminal helix (helix H10) that can insert into the crevice, further contributing to its shape and binding capabilities .

What are the primary physiological roles of NCS-1 in human cells?

NCS-1 participates in numerous critical neuronal functions, including:

• Regulation of Ca2+ channels

• Control of exocytosis mechanisms

• Promotion of synaptogenesis

• Facilitation of axonal growth

• Enhancement of learning and memory processes

• Provision of neuroprotection

• Support for axonal regeneration

The multifunctionality of NCS-1 stems from its ability to recognize and regulate different unrelated target proteins, including G-protein-coupled receptors (GPCRs) and their regulators, Ca2+ channels, guanine nucleotide exchange factors (GEF), and various kinases. These interactions can occur in both Ca2+-dependent and Ca2+-independent manners .

What are the most effective methods for studying NCS-1 protein-protein interactions?

Based on the research data, several effective methodologies have been employed to study NCS-1 protein-protein interactions:

• Co-immunoprecipitation assays: These have been successfully used to determine interaction affinities between NCS-1 and various binding partners, such as Ric-8A. This technique helps identify which regions of partner proteins are involved in the interaction .

• Crystallographic studies: X-ray crystallography with peptides of different lengths has been instrumental in identifying regions necessary and sufficient for NCS-1 recognition. This approach has revealed significant conformational changes that occur during binding interactions .

• Cell-based protein-protein interaction assays: These assays have been used to validate interactions observed in vitro and to test the effects of mutations on binding affinity .

• Reporter assay systems: These have been developed to investigate transcriptional regulation, such as the effect of valproic acid (VPA) and GSK3 activity on NCS-1 gene transcription .

A combination of these approaches provides comprehensive insights into NCS-1's interaction landscape.

How can I express and purify recombinant human NCS-1 for structural studies?

While the provided search results don't include a specific protocol for NCS-1 purification, based on the crystallographic studies mentioned, a typical approach would include:

• Cloning: Insert the full-length human NCS-1 cDNA into a suitable expression vector with an affinity tag (His-tag or GST-tag).

• Expression system: Use either bacterial (E. coli) or eukaryotic (insect cells) expression systems depending on the need for post-translational modifications.

• Purification steps:

  • Affinity chromatography (using the tag)

  • Ion-exchange chromatography

  • Size-exclusion chromatography for final polishing

• Quality control: Verify purified protein using SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and integrity.

• Functional validation: Test calcium binding capacity using circular dichroism or fluorescence spectroscopy.

For crystallography studies specifically, it's important to maintain high protein concentration (typically >5 mg/ml) and ensure buffer conditions that promote protein stability.

How is NCS-1 implicated in neuropsychiatric disorders?

NCS-1 has been implicated in several psychiatric and neurological conditions:

• X-linked mental retardation and autism: Alterations in NCS-1 expression or function may contribute to neurodevelopmental disorders, affecting synapse formation and neuronal connectivity .

• Schizophrenia: NCS-1 dysregulation has been observed in schizophrenia patients, potentially affecting dopaminergic signaling, which is crucial in this disorder .

• Bipolar disorder: Research indicates that NCS-1 may be affected by mood stabilizers like valproic acid (VPA). Studies show that VPA upregulates NCS-1 expression through inhibition of glycogen synthase kinase 3 (GSK3), which may contribute to its therapeutic effects .

The multifunctional nature of NCS-1 and its involvement in regulating Ca2+ channels and neurotransmitter release makes it a critical factor in conditions where synaptic transmission and neuronal excitability are disrupted .

What experimental evidence supports NCS-1's role in anxiety-related behaviors?

Research shows that NCS-1 may play a significant role in mediating anxiety-related behaviors:

• Viral vector-mediated expression: Studies using adenoassociated viruses (AAV) to overexpress NCS-1 in the dorsomedial frontal cortex of mice demonstrated anxiolytic-like effects. These mice showed increased exploration in anxiety-provoking situations .

• Behavioral assessments: Mice with upregulated NCS-1 expression in the dorsomedial frontal cortex displayed:

  • Reduced anxiety-like behaviors

  • Enhanced social interaction

  • Greater preference for social stimuli over non-social stimuli

• Quantifiable differences: When tested, NCS1-EYFP mice showed a significantly higher time ratio between social and non-social chambers (1.87 ± 0.17) compared to control EYFP mice (1.33 ± 0.06) .

These findings suggest that targeted upregulation of NCS-1 in specific brain regions might have therapeutic potential for anxiety disorders.

How do post-translational modifications affect NCS-1 function and binding properties?

While the search results don't directly address post-translational modifications of NCS-1 itself, they provide insights into how NCS-1 affects the phosphorylation of binding partners. For example:

• Phosphorylation regulation: NCS-1 interacts with Ric-8A, which undergoes phosphorylation at S435 and T440 (human S436 and T441). This phosphorylation allows Ric-8A to attach to positively charged patches, affecting its interaction with G proteins .

• Conformational changes: Binding of phosphorylated Ric-8A (pRic-8A) to Gα does not substantially alter the conformation of the ARM-HEAT repeat domain, but significant conformational changes occur in the HEAT repeat 9 region that interacts with NCS-1 .

Future research should investigate:

• Whether NCS-1 itself undergoes phosphorylation, acetylation, or other modifications

• How these modifications might affect calcium binding properties

• Whether modifications alter NCS-1's subcellular localization or protein-protein interactions

What is the regulatory mechanism of NCS-1 expression in neurons?

Based on the research data, NCS-1 expression is regulated by several mechanisms:

• Transcriptional regulation: A 2,005 bp nucleotide sequence from the human NCS-1 promoter contains TATA boxes, GpC islands, and numerous DNA binding domains for transcription factors including c-Ets, Creb, and AP1 .

• GSK3-mediated regulation: Glycogen synthase kinase 3 (GSK3) appears to be a key regulator of NCS-1 expression. Inhibition of GSK3 by valproic acid (VPA) or specific inhibitors like TDZD-8 leads to increased NCS-1 mRNA and protein levels .

• Signaling pathway involvement: The regulation involves a pathway where:

  • VPA activates brain Akt

  • Activated Akt phosphorylates GSK3α and GSK3β, leading to their inhibition

  • This modulation requires beta-arrestin-2 (βArr2), a signaling adaptor molecule that mediates G-protein coupled receptor (GPCR) desensitization and signaling

  • Dopamine D2 receptors, which signal via βArr2 to inactivate Akt, also play a role in this regulatory mechanism

This complex regulatory network suggests that NCS-1 expression is tightly controlled and responsive to various signaling inputs in neurons.

What techniques can be used to study the dynamic calcium-dependent conformational changes in NCS-1?

To study the calcium-dependent conformational changes in NCS-1, researchers could employ the following techniques:

• X-ray crystallography: This technique has been successfully used to determine the structure of NCS-1 bound to peptides from its binding partners, revealing conformational changes that occur upon binding .

• Nuclear Magnetic Resonance (NMR) spectroscopy: This would be particularly useful for studying dynamic conformational changes in solution, providing insights into the flexibility of different regions of NCS-1 upon calcium binding.

• Fluorescence resonance energy transfer (FRET): By introducing fluorescent tags at strategic positions, researchers can monitor distance changes in real-time as calcium binds to NCS-1.

• Circular dichroism (CD) spectroscopy: This can provide information about changes in secondary structure content upon calcium binding.

• Molecular dynamics simulations: Computational approaches can model conformational changes and binding interactions at an atomic level, complementing experimental data.

These methods, used in combination, would provide comprehensive insights into how calcium binding affects NCS-1 structure and subsequently its interactions with various binding partners.

What is known about the NCS-1 and Ric-8A interaction interface?

The interaction between NCS-1 and Ric-8A has been extensively characterized at the structural level:

• Interaction site on Ric-8A: The HEAT repeat 9 of the ARM/HEAT repeat domain of Ric-8A, composed of a two-helix bundle (called a9 and b9), is the primary site of interaction with NCS-1. Specifically, when Ric-8A binds to NCS-1, helix a9 unfolds while helix b9 refolds, resulting in two regions named R1 and R2, with a changed relative orientation .

• Binding determinants:

  • The R1-R2 loop, formed by residues T410, G411, Y412, and G413, contributes significantly to the protein-protein interaction, providing three hydrogen bonds.

  • These four residues account for 28% of total contacts between human NCS-1 and Ric-8A .

• Minimal sufficient region: Crystal structures and protein-protein interaction assays identified that the region of Ric-8A necessary and sufficient for NCS-1 recognition is conserved between human and rat proteins. This region corresponds to the two-helix bundle of HEAT repeat 9 and a disordered region that forms an extended coil when phosphorylated .

• Competition with G proteins: NCS-1 and G-proteins compete for Ric-8A binding, suggesting they may share certain interaction surfaces. This competitive binding is likely important for regulating G-protein signaling pathways .

How does calcium binding modulate NCS-1's interaction with different partners?

Calcium binding to NCS-1 induces structural rearrangements that affect its interactions with target proteins:

Understanding these calcium-dependent mechanisms provides insights into how NCS-1 can interact with multiple partners and participate in diverse cellular functions.

How do mood stabilizers like valproic acid affect NCS-1 expression and function?

Valproic acid (VPA), a mood stabilizer used for managing bipolar disorder, significantly affects NCS-1 expression through several mechanisms:

• Upregulation effect: VPA administration leads to increased NCS-1 mRNA and protein levels in both cell lines and the mouse frontal cortex .

• Mechanism of action:

  • VPA activates brain Akt

  • Activated Akt phosphorylates and inhibits glycogen synthase kinase 3 (GSK3α and GSK3β)

  • This inhibition of GSK3 results in upregulation of NCS-1

• Signaling pathway requirements:

  • The modulation of Akt and GSK3 activity by VPA requires the signaling adaptor molecule beta-arrestin-2 (βArr2)

  • Dopamine D2 receptors (D2R), which signal via βArr2 to inactivate Akt, play a role in the regulation of GSK3 by VPA

• Behavioral effects: Upregulation of NCS-1 in the dorsomedial frontal cortex promotes anxiolytic-like and pro-social behaviors in mice, suggesting that some of VPA's therapeutic effects may be mediated through NCS-1 upregulation .

What experimental approaches can be used to target NCS-1 for therapeutic purposes?

Based on the research data, several experimental approaches could be employed to target NCS-1 for therapeutic purposes:

• Viral vector-mediated gene therapy: Adenoassociated viruses (AAV) expressing NCS-1 have been successfully used to upregulate NCS-1 in specific brain regions, leading to anxiolytic-like and pro-social behaviors in mice. This region-specific approach could be refined for targeted therapeutic applications .

• Small molecule modulators: Compounds that mimic the effects of valproic acid on GSK3 inhibition could potentially upregulate NCS-1 expression with fewer side effects. The data on VPA's mechanism of action provides a foundation for developing more specific modulators .

• Peptide-based approaches: The detailed structural information on NCS-1's interaction with partners like Ric-8A could be leveraged to design peptides or peptidomimetics that modulate specific NCS-1 interactions while preserving others .

• Promoter-targeted approaches: The identification of the NCS-1 promoter region and its response elements provides potential targets for transcriptional regulation of NCS-1 expression. Compounds that activate specific transcription factors binding to this region could selectively enhance NCS-1 expression .

For any therapeutic approach, it would be crucial to consider:

• Region-specific effects in the brain

• Potential off-target effects due to NCS-1's multiple binding partners

• Dosage and timing to achieve optimal modulation without disrupting normal signaling

What gaps remain in our understanding of human NCS-1 compared to other species?

Several important knowledge gaps remain regarding human NCS-1 compared to other species:

• Species-specific interactions: While some binding partners like Ric-8A show conservation between rat and human systems, comprehensive comparative studies of the full interaction networks across species are lacking. This is critical as subtle differences may have significant functional implications .

• Tissue-specific expression patterns: More detailed mapping of human NCS-1 expression across different tissues and cell types would enhance our understanding of its diverse roles beyond the nervous system.

• Disease-specific variants: Further investigation into human-specific NCS-1 variants associated with neuropsychiatric disorders would provide valuable insights into pathological mechanisms.

• Developmental regulation: Studies on the developmental regulation of NCS-1 expression in humans compared to model organisms would help clarify its role in neurodevelopment and potentially in neurodevelopmental disorders.

• Compensatory mechanisms: Better understanding of redundancy and compensation within the NCS family in humans versus other species would help explain why some NCS-1-related phenotypes differ across species.

Future studies should address these gaps to better translate findings from model organisms to human health applications.

How can computational approaches enhance our understanding of NCS-1 function?

Computational approaches offer powerful tools to advance our understanding of NCS-1 function:

• Molecular dynamics simulations: These can model the conformational dynamics of NCS-1 upon calcium binding and interaction with various partners, providing insights difficult to obtain experimentally. Simulations could reveal transient conformational states important for target recognition.

• Systems biology models: Integrating NCS-1 into broader signaling networks through computational modeling could help predict how perturbations in NCS-1 expression or function might affect downstream pathways. This would be particularly valuable for understanding its role in complex disorders like schizophrenia and bipolar disorder.

• Machine learning approaches: Using machine learning to analyze patterns in NCS-1 binding partners could help identify common structural or sequence features that determine binding specificity, potentially leading to the prediction of novel interaction partners.

• Genomic data integration: Analysis of genomic and transcriptomic data across human populations could identify regulatory variants affecting NCS-1 expression and potential associations with disease susceptibility.

• Drug discovery applications: Virtual screening and structure-based drug design could identify compounds that modulate NCS-1 function or specific protein-protein interactions, potentially leading to novel therapeutic approaches.

These computational approaches, when combined with experimental validation, would significantly accelerate our understanding of NCS-1 function and its therapeutic potential.