NOS1AP Antibody

What is NOS1AP and what are its primary functions in cellular biology?

NOS1AP (Nitric Oxide Synthase 1 Adaptor Protein, previously known as CAPON) is a scaffolding protein that serves as an adapter involved in neuronal nitric oxide (NO) synthesis regulation through its association with neuronal nitric oxide synthase (nNOS/NOS1) . The protein mediates signaling within a complex that includes the NMDA receptor, PSD-95, and nNOS . NOS1AP competes with PSD-93/95 for binding to nNOS, and excessive NOS1AP can inhibit the interaction between PSD-93/95 and nNOS, thereby affecting the activation of NO synthase through NMDA receptors in response to calcium influx . Additionally, NOS1AP participates in the mitogen-activated protein kinase signaling cascade and Hippo signaling pathway through interactions with Dexras 1 and Scribble, respectively .

What is the structural composition of NOS1AP protein?

NOS1AP contains 506 amino acid residues with a molecular mass of approximately 56.2 kDa . The protein possesses two primary protein interaction domains: an N-terminal phosphotyrosine binding domain (PTB) and a C-terminal PDZ binding domain (PDZ-BD) through which it interacts with nitric oxide synthase 1 (NOS1) . Multiple alternative transcripts of NOS1AP have been described, which encode the same N-terminal domains (including the PTB domain) but distinct C-terminal domains lacking the PDZ-BD . The canonical protein is reported to have subcellular localization in cell projections .

In which tissues and cell types is NOS1AP predominantly expressed?

NOS1AP is expressed in multiple tissues including the heart, pancreas, skeletal muscles, and nervous system . Within the kidney, single-cell mRNA sequencing data from adult mouse glomeruli and adult human kidney experiments demonstrate that NOS1AP mRNA transcript is predominantly expressed in podocytes relative to other kidney glomerular and renal epithelial cell types . Immunofluorescence microscopy in adult rat glomeruli has revealed Nos1ap staining in a linear pattern that shows partial colocalization with the podocyte slit diaphragm marker nephrin, consistent with podocyte foot process localization .

What are the key considerations when selecting a NOS1AP antibody for research applications?

When selecting a NOS1AP antibody, researchers should consider several critical factors: (1) The specific epitope recognized by the antibody, as NOS1AP has multiple isoforms due to alternative splicing with three reported isoforms in humans ; (2) The validation methods used by the manufacturer, with preference given to antibodies validated through multiple techniques such as Western blot, immunofluorescence, and knockout/knockdown controls; (3) The species reactivity, as there may be sequence variations between human, mouse, and rat NOS1AP; and (4) The specific application requirements, as antibodies optimized for Western blotting may not perform equally well in immunohistochemistry or immunoprecipitation experiments . Additionally, researchers should verify whether the antibody recognizes the full-length protein or specific domains, particularly when studying specific isoforms or truncated variants.

How can researchers validate NOS1AP antibody specificity for their experiments?

Validation of NOS1AP antibody specificity is essential to ensure experimental reliability. A comprehensive validation approach includes: (1) Testing the antibody against overexpressed tagged NOS1AP protein in immunoblotting and immunofluorescence studies to confirm recognition of the target protein ; (2) Performing antibody preabsorption tests with the immunogen to demonstrate signal abrogation, as demonstrated in studies where Nos1ap signal was eliminated upon preabsorption of the antibody with its immunogen ; (3) Using knockout/knockdown models as negative controls; (4) Confirming expected subcellular localization patterns, such as the localization to F-actin containing filopodia and podosomes in podocytes ; and (5) Performing comparative analyses with multiple antibodies targeting different epitopes of NOS1AP to confirm consistent detection patterns.

What are the optimal protocols for detecting NOS1AP in different subcellular compartments?

For comprehensive detection of NOS1AP in different subcellular compartments, researchers should employ targeted protocols tailored to the protein's distinct localizations. For membrane and cytoskeletal-associated NOS1AP, immunofluorescence microscopy with appropriate fixation is crucial. Research has shown that in 24% of immortalized human podocytes, NOS1AP localizes to F-actin containing filopodia, while in 44% of cells, it colocalizes with F-actin in peripheral ring structures identified as podosomes through NWASP colocalization . The remaining 32% exhibit diffuse localization.

Optimal protocols should include: (1) Paraformaldehyde fixation (4%) followed by permeabilization with 0.1% Triton X-100 to preserve cytoskeletal structures; (2) Co-staining with markers such as phalloidin for F-actin, NWASP for podosomes, and compartment-specific markers depending on the cell type investigated; (3) Super-resolution microscopy techniques (such as STED or STORM) for detailed localization studies, particularly for structures like filopodia and podosomes; and (4) Live-cell imaging with GFP-tagged NOS1AP to observe dynamic localization patterns in real-time, especially when studying filopodia formation processes .

How can NOS1AP antibodies be used to investigate protein-protein interactions in neuropsychiatric disorders?

NOS1AP antibodies serve as valuable tools for investigating protein-protein interactions implicated in neuropsychiatric disorders through several methodological approaches: (1) Co-immunoprecipitation experiments to identify interacting partners, particularly with nNOS, PSD-95, and α-synuclein, which has been shown to interact with NOS1AP in synucleinopathy models ; (2) Proximity ligation assays (PLA) to visualize and quantify in situ protein interactions in brain tissue samples from patients with schizophrenia, depression, or other neuropsychiatric conditions where NOS1AP levels are altered ; (3) Immunohistochemistry of post-mortem brain sections from psychiatric disorder patients to examine NOS1AP expression and localization patterns, as research has shown elevated expression of the short NOS1AP isoform in postmortem brain samples of patients with schizophrenia ; and (4) Combined use of NOS1AP antibodies with neuron-specific markers in induced pluripotent stem cell (iPSC)-derived neuronal models from patients to investigate disease-specific alterations in NOS1AP interactions.

What methodological approaches can be used to study NOS1AP aggregation properties?

Research has demonstrated that NOS1AP can form aggregates under certain conditions, requiring specific methodological approaches to investigate this phenomenon . To study NOS1AP aggregation, researchers can employ: (1) Sedimentation assays and detergent resistance tests to isolate and characterize NOS1AP aggregates, as studies have shown NOS1AP forms SDS-resistant aggregates in vitro ; (2) Fluorescence microscopy with Congo Red staining combined with cross-polarized light examination to determine if aggregates are amyloid in nature (research indicates NOS1AP forms non-amyloid aggregates) ; (3) Electron microscopy to characterize the morphology of NOS1AP aggregates, which have been observed to form amorphous rather than fibrillar structures ; (4) Heterologous expression systems such as bacterial and yeast models for initial screening of aggregation propensity; and (5) Co-aggregation studies with proteins like α-synuclein to investigate potential roles in synucleinopathies, as bioinformatic analyses have revealed NOS1AP as an aggregation-prone protein interacting with α-synuclein .

How does NOS1AP expression relate to podocyte function and kidney disease pathogenesis?

Recent research has revealed a critical role for NOS1AP in podocyte function and kidney disease pathogenesis . Studies demonstrate that recessive NOS1AP variants impair actin remodeling in podocytes and cause glomerular disease in humans, kidney organoids, and mice . NOS1AP is predominantly expressed in mammalian podocytes relative to other kidney glomerular and renal epithelial cell types, and it localizes to podocyte foot processes, showing partial colocalization with the slit diaphragm marker nephrin .

Functionally, wild-type NOS1AP, but not patient-derived mutants, promotes filopodia formation in podocytes, similar to the function of known nephrotic syndrome genes CDC42, ITSN1, and ITSN2 . This process appears to be mediated through CDC42 activation, as NOS1AP overexpression increases active CDC42 levels and promotes filopodia and podosome formation, while pharmacologic inhibition of CDC42 or its effector formin proteins reduces NOS1AP-induced filopodia formation . In NOS1AP knockdown podocytes, the reduced podocyte migration rate can be rescued by overexpression of wild-type NOS1AP but not by constructs bearing patient variants, or alternatively by constitutively active CDC42 Q61L or the formin DIAPH3 . These findings establish a mechanistic link between NOS1AP function, actin cytoskeletal dynamics, and podocyte biology in kidney disease.

What is the evidence linking NOS1AP to neuropsychiatric disorders?

Substantial evidence connects NOS1AP to various neuropsychiatric disorders . Elevated NOS1AP mRNA and protein levels have been found in the blood and dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia . Increased NOS1AP immunoreactivity has been observed in the DLPFC and anterior cingulate cortex of patients with major depressive disorder, and increased NOS1AP mRNA in the hippocampus of schizophrenia patients .

Genetic studies have established that NOS1AP variants are associated with schizophrenia endophenotypes and depression-related traits in schizophrenia patients . Additionally, NOS1AP variants have been linked to symptom severity and depression and anxiety symptoms in posttraumatic stress disorder (PTSD) . The expression of the short NOS1AP isoform is elevated in postmortem brain samples from schizophrenia patients, and the protein levels of different NOS1AP isoforms are increased in Brodmann area 46 of the frontal cortex in these patients .

Preclinical studies support these clinical observations, demonstrating that overexpression of murine NOS1AP in the mouse dentate gyrus produces anxiogenic effects, while downregulation of NOS1AP in the medial prefrontal cortex reverses stress-induced depression-like behavior in mice . At the cellular level, NOS1AP overexpression in cultured neurons reduces dendritic growth and the number of mature dendritic spines while increasing filopodia-like protrusions , resembling observations in post-mortem studies of various mental conditions including schizophrenia, mood disorders, and intellectual disability.

How does NOS1AP interact with α-synuclein and what are the implications for neurodegenerative diseases?

Recent research has uncovered a previously unknown interaction between NOS1AP and α-synuclein, suggesting potential implications for synucleinopathies like Parkinson's disease . Bioinformatic analysis identified NOS1AP as an aggregation-prone protein that interacts with α-synuclein . While the precise molecular mechanisms of this interaction remain under investigation, the finding suggests that NOS1AP may play a role in the development or progression of synucleinopathies .

This interaction could be particularly significant given that NOS1AP forms detergent-resistant non-amyloid aggregates when overproduced . The protein was found to form SDS-resistant aggregates immediately after dilution from denaturing conditions, with these aggregates remaining stable for at least 24 hours of incubation at 37°C . Importantly, these aggregates are non-amyloid in nature, as demonstrated by the absence of characteristic apple-green birefringence under cross-polarized light following Congo Red staining . Electron microscopy confirmed that NOS1AP forms amorphous rather than fibrillar aggregates .

The interaction between NOS1AP and α-synuclein, combined with their aggregation properties, suggests potential cooperative or competitive effects in protein aggregation processes implicated in neurodegenerative diseases. This represents a novel area for therapeutic exploration, particularly for synucleinopathies where protein aggregation plays a central pathogenic role.

How can NOS1AP antibodies be used in combination with genetic approaches to understand disease mechanisms?

Integrating NOS1AP antibodies with genetic approaches offers powerful strategies for elucidating disease mechanisms . Researchers can employ: (1) CRISPR/Cas9 gene editing to introduce disease-associated NOS1AP variants in cellular or animal models, followed by antibody-based detection of altered expression, localization, or interaction patterns; (2) Human kidney organoid models harboring NOS1AP patient variants, which have revealed malformed glomeruli with increased apoptosis ; (3) Studies of Nos1ap homozygous mutant mice that, while viable through adulthood without reported renal phenotypes, are prone to cardiac arrhythmias and dysfunction when challenged with oxidative stress-inducing agents ; and (4) Combined transcriptomic and proteomic analyses with NOS1AP antibody-based immunoprecipitation to identify novel interacting partners and signaling pathways affected by disease-associated variants.

This integrated approach has already yielded significant insights, such as the discovery that recessive variants in NOS1AP cause early-onset nephrotic syndrome in humans, with corresponding glomerular abnormalities in kidney organoids and severe proteinuria and podocyte foot process effacement in mice with biallelic variants in Nos1ap .

What methodological challenges exist when studying post-translational modifications of NOS1AP?

Investigating post-translational modifications (PTMs) of NOS1AP presents several methodological challenges that researchers must address: (1) The availability of modification-specific antibodies for NOS1AP is limited, requiring careful validation of any PTM-specific antibodies; (2) The dynamic nature of many PTMs necessitates time-course studies with precise cellular stimulation protocols to capture transient modifications; (3) The presence of multiple isoforms of NOS1AP due to alternative splicing complicates the interpretation of PTM patterns, as different isoforms may undergo distinct modifications ; and (4) The subcellular localization of NOS1AP to multiple compartments, including filopodia, podosomes, and diffuse cytoplasmic distribution , requires compartment-specific enrichment strategies to study location-specific modifications.

To overcome these challenges, researchers should consider employing mass spectrometry-based proteomics following immunoprecipitation with pan-NOS1AP antibodies, combining subcellular fractionation with phospho-specific Western blotting, and utilizing proximity labeling approaches like BioID or APEX to identify context-specific PTM regulators of NOS1AP.

How can advanced imaging techniques be optimized for studying NOS1AP localization dynamics in live cells?

Advanced imaging techniques offer powerful approaches for investigating NOS1AP localization dynamics, particularly important given its roles in actin remodeling and filopodia formation . Optimization strategies include: (1) Generation of fluorescent protein-tagged NOS1AP constructs (GFP-NOS1AP) that maintain physiological function, as demonstrated in studies where GFP-tagged wild-type NOS1AP overexpression increased filopodia formation in 59% of GFP-positive podocytes compared to only 1% in control cells ; (2) Implementation of live-cell super-resolution microscopy techniques such as Structured Illumination Microscopy (SIM) or Lattice Light-Sheet Microscopy to capture dynamic changes in NOS1AP localization with minimal phototoxicity; (3) Development of FRET-based biosensors to monitor NOS1AP interactions with binding partners like nNOS or CDC42 in real-time; and (4) Correlative light and electron microscopy (CLEM) to connect dynamic fluorescence imaging with ultrastructural context, particularly valuable for studying NOS1AP's association with specialized actin structures.

These advanced imaging approaches can reveal how disease-associated mutations affect the spatiotemporal dynamics of NOS1AP localization and interactions, providing mechanistic insights into pathological processes.