NCS1 Human

What is NCS1 and what is its primary function in human neurons?

NCS1 is a highly conserved calcium binding protein abundantly expressed in neurons. It functions primarily as a modulator of intracellular calcium homeostasis, calcium-dependent signaling pathways, as well as neuronal transmission and plasticity . Unlike other neuronal calcium sensors, NCS1 is found outside the nervous system and does not contain a Ca²⁺/Myr switch, causing it to be constantly bound to the membrane .

The protein structure of NCS1 consists of two pairs of EF-hand motifs, with three functional calcium-binding sites: EF-2, EF-3, and EF-4. EF-2 and EF-3 can also recognize Mg²⁺ and serve as structural sites that allow the protein to adopt its tertiary fold. Meanwhile, EF-4 functions as a regulatory Ca²⁺ binding site capable of sensing changes in cytosolic calcium levels in neurons .

Methodologically, researchers can study NCS1 function through calcium imaging techniques, electrophysiology, and protein-protein interaction assays to understand how it modulates neuronal activity and signaling pathways.

What experimental models are available for studying NCS1 function in humans?

Several experimental models exist for studying NCS1 function in humans, with CRISPR-Cas9 genome editing being particularly valuable. Researchers have successfully generated NCS1 knockout human induced pluripotent stem cell (hiPSC) lines that show regular expression of pluripotent markers, normal iPSC morphology and karyotype, with no detectable off-target effects on potentially affected genes .

These knockout cell lines serve as valuable tools for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological diseases . The methodology for creating such models typically involves:

• Design of guide RNAs targeting specific regions of the NCS1 gene

• Transfection of hiPSCs with CRISPR-Cas9 components

• Clonal selection and expansion

• Validation through sequencing and expression analysis

• Functional characterization through calcium imaging and electrophysiology

Additionally, researchers can differentiate these hiPSCs into neurons to study the effects of NCS1 knockout on neuronal development, synapse formation, and electrophysiological properties.

What protein interactions does NCS1 participate in?

NCS1 participates in a wide range of protein interactions due to its ability to recognize and regulate different target proteins. The multifunctionality of NCS1 relies on its ability to interact with G-protein-coupled receptors (GPCRs) and some of their regulators, Ca²⁺ channels, guanine nucleotide exchange factors (GEF), and kinases, both in a Ca²⁺-dependent and -independent manner .

One well-characterized interaction is between NCS1 and Ric-8A, where they coregulate synapse number and probability of neurotransmitter release . NCS1 uses a surface-exposed hydrophobic crevice to recognize its targets, which generally present short helical motifs that bind to the N- or C-terminal part of this large cavity. The shape and size of this hydrophobic crevice, along with hydrophilic residues at its border, determine target specificity .

Methodological approaches to study these interactions include:

• Co-immunoprecipitation assays

• Crystal structure analysis

• Fluorescence resonance energy transfer (FRET)

• Surface plasmon resonance (SPR)

• Yeast two-hybrid screening

How does NCS1 contribute to normal neuronal function?

NCS1 participates in a wide range of important neuronal functions that collectively impact neural circuit development and function. It serves as a regulator of Ca²⁺ channels, which are critical for controlling neuronal excitability and neurotransmitter release . Additionally, NCS1 regulates exocytosis, the process by which neurotransmitters are released into the synaptic cleft.

Beyond its role in neurotransmission, NCS1 is involved in synaptogenesis (the formation of synapses between neurons) and axonal growth . These developmental functions ultimately affect higher cognitive processes such as learning and memory. Research has also revealed roles for NCS1 in neuroprotection and axonal regeneration, suggesting it contributes to neuronal resilience and recovery after injury .

Methodologically, these functions can be studied through:

• Electrophysiological recordings to measure synaptic transmission

• Live imaging to track synapse formation and axonal growth

• Calcium imaging to visualize calcium dynamics in neurons

• Behavioral assays in animal models with altered NCS1 expression

What is known about NCS1's role in neuropsychiatric disorders?

NCS1 has been implicated in several pathological processes related to neuropsychiatric disorders. Research has linked NCS1 to X-linked mental retardation and autism, schizophrenia, and bipolar disorder . These associations suggest that dysregulation of calcium signaling through NCS1 may contribute to the pathophysiology of these conditions.

The generation of NCS1 knockout human induced pluripotent stem cell lines provides a valuable tool for studying these relationships . These cell lines can be differentiated into neurons to investigate how the absence of NCS1 affects neuronal development, connectivity, and function in the context of these disorders.

Methodological approaches to studying NCS1's role in neuropsychiatric disorders include:

• Gene association studies in patient populations

• Expression analysis in post-mortem brain tissue

• Functional studies using knockout or knockdown models

• Electrophysiological and calcium imaging analyses in patient-derived neurons

• Behavioral assessments in animal models with altered NCS1 expression

What structural mechanisms determine NCS1 binding specificity to target proteins?

The structural determinants of NCS1 target specificity are based on the shape and size of its hydrophobic crevice. NCS1 contains a dynamic C-terminal helix (helix H10) that can insert into the crevice, thus contributing to its shape . Since Ca²⁺ binding promotes structural rearrangements, the occupancy of the three Ca²⁺ binding sites also determines affinity for protein partners .

The structures of several NCS proteins bound to their corresponding targets have shown that these Ca²⁺ sensors use a surface-exposed hydrophobic crevice to recognize their targets, which generally present short helical motifs that bind to the N- or C-terminal part of this large cavity . Additionally, the presence of hydrophilic residues at the border of the crevice contributes to target specificity and constitutes hot spots for interactions with different targets .

Methodologically, researchers can study these mechanisms through:

• X-ray crystallography of NCS1-target complexes

• Site-directed mutagenesis of key residues

• Isothermal titration calorimetry to measure binding affinities

• Molecular dynamics simulations to observe conformational changes

• Nuclear magnetic resonance spectroscopy to study dynamics in solution

How does the interaction between NCS1 and Ric-8A regulate G-protein signaling?

The interaction between NCS1 and Ric-8A has been extensively studied, revealing important structural and functional insights. Crystal structure analysis identified that the region of Ric-8A necessary and sufficient for NCS1 recognition corresponds to the two-helix bundle that constitutes the HEAT repeat 9 of the ARM-HEAT repeat domain .

When binding to NCS1, the HEAT repeat 9 of Ric-8A undergoes a substantial conformational change: helix a9 unfolds while helix b9 refolds, resulting in regions named R1 and R2 . This interaction is functionally significant as NCS1 and G-proteins compete for Ric-8A binding, suggesting they could share certain interaction surfaces .

Through a combination of crystallographic work using Ric-8A peptides of different lengths and cell-based protein-protein interaction assays with various Ric-8A and NCS1 mutants, researchers have identified specific regions necessary for this interaction . Interestingly, a C-terminally truncated construct of human Ric-8A (hRic-8A-424) showed significantly higher affinity for NCS1 compared to full-length hRic-8A, suggesting that the HEAT repeat 9, but not the rH10 or rH11 helices, is implicated in the protein-protein interaction .

Methodological approaches to study this interaction include:

• Co-immunoprecipitation assays with different protein constructs

• Crystal structure analysis of protein complexes

• Mutational analysis of binding interfaces

• Functional assays measuring G-protein activation

• Live-cell imaging of protein interactions

What is the role of NCS1 in cancer immunotherapy and prognosis?

Recent research has highlighted NCS1's involvement in immune regulation and its potential use as a prognostic biomarker for multiple cancer types, including SKCM (skin cutaneous melanoma), LIHC (liver hepatocellular carcinoma), BRCA (breast cancer), COAD (colon adenocarcinoma), and KIRC (kidney renal clear cell carcinoma) .

Researchers have employed multiple methodological approaches to study NCS1's role in cancer:

• Analysis of RNA sequencing expression data from TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) programs using tools like GEPIA 2.0

• Assessment of NCS1's correlation with clinicopathological features using Kaplan-Meier Plotter and The Human Protein Atlas

• Evaluation of NCS1 DNA promoter region methylation in pan-cancer data using the UALCAN database

• Immune score assessment using the "immuneeconv" R package, which integrates six algorithms: TIMER, xCell, MCP-counter, CIBERSORT, EPIC, and stromal score

Through these approaches, researchers have analyzed the association between NCS1 and immunosuppressive, immunostimulatory, and MHC molecules, as well as tumor-infiltrating lymphocytes (TILs) and chemokines regulating T-cell trafficking in pan-cancer . This research direction provides valuable insights for developing new therapeutic approaches combining NCS1-targeted treatments with immunotherapy.

How can single-cell analysis techniques advance understanding of NCS1 function?

While not explicitly discussed in the search results, single-cell analysis techniques represent a cutting-edge methodological approach for studying NCS1 function in heterogeneous cell populations. These techniques can overcome limitations of bulk tissue analysis, which may mask cell type-specific expression patterns and functions of NCS1.

Methodological approaches include:

• Single-cell RNA sequencing (scRNA-seq) to profile NCS1 expression across different cell types and correlate it with expression of other genes involved in calcium signaling, synaptic function, and disease pathways

• Single-cell proteomics to analyze NCS1 protein levels and post-translational modifications

• Spatial transcriptomics to map NCS1 expression within tissue architecture

• Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to simultaneously analyze NCS1 expression and cell surface protein markers

• Patch-seq, which combines electrophysiological recording with single-cell transcriptomics to link NCS1 expression to functional properties

These approaches can reveal cell type-specific roles of NCS1 in both normal physiology and disease states, potentially identifying new therapeutic targets for conditions associated with NCS1 dysfunction.

What methodologies are most effective for studying NCS1 phosphorylation and its functional consequences?

Phosphorylation of proteins like NCS1 can significantly alter their binding properties and functions. While the search results don't specifically address NCS1 phosphorylation, they do mention phosphorylated Ric-8A (pRic-8A) and its interaction with Gα . This suggests that phosphorylation states are important in the signaling pathways involving NCS1 and its binding partners.

Effective methodological approaches for studying NCS1 phosphorylation include:

• Mass spectrometry-based phosphoproteomics to identify phosphorylation sites

• Phospho-specific antibodies for Western blotting and immunoprecipitation

• Phosphomimetic and phospho-deficient mutants to study functional consequences

• In vitro kinase assays to identify kinases responsible for NCS1 phosphorylation

• Live-cell imaging using phospho-sensors to monitor dynamics of phosphorylation

• Structural studies comparing phosphorylated and non-phosphorylated forms

Understanding the phosphorylation status of NCS1 in different cellular contexts could provide insights into its regulation and function in health and disease.