Nck-associated Protein 1 Research Articles

NCK-associated protein 1 (NCKAP1) And Angiopoietin-like protein 1 (ANGPTL1): A novel prognostic markers and promising therapeutic target for colorectal carcinoma patients

NCK-associated protein 1 (NCKAP1) And Angiopoietin-like protein 1 (ANGPTL1): A novel prognostic markers and promising therapeutic target for colorectal carcinoma patients

A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

NCKAP1 as a prognostic and immunological biomarker: pan-cancer analysis and validation in renal clear cell carcinoma.

To systematically investigate the expression, prognostic value, genetic alterations, immune infiltration, and molecular function of Nck-associated protein 1 (NCKAP1) in a pan-cancer analysis, with a specific focus on its association with kidney renal cell carcinoma (KIRC). We analyzed the role of NCKAP1 across various tumor types using data from The Cancer Genome Atlas (TCGA). The Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) database was used to assess the correlation between NCKAP1 expression levels and overall survival (OS) and disease-free survival (DFS) across different cancers, as well as its association with cancer stage. Genetic alterations of NCKAP1 were explored using CBioPortal, and their prognostic implications were assessed. NCKAP1 was further analyzed through Gene Ontology and protein interaction network analyses. Immunohistochemistry (IHC) staining from the Human Protein Atlas (HPA) database evaluated NCKAP1 levels in KIRC tissues. Functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays, were conducted to determine the effects of NCKAP1 overexpression on cell growth rate and their ability to invade, proliferate, migrate in a KIRC (786-O) cell line. The relationship between NCKAP1 expression and immune infiltration in KIRC was systematically examined using the Tumor Immune Estimation Resource. NCKAP1 expression was significantly altered in most tumor types compared to corresponding non-tumor tissues. Survival analysis indicated that low NCKAP1 expression was associated with poor OS, DFS, and advanced cancer stage (P < 0.05) specifically in KIRC. Genetic alterations in NCKAP1 were linked to clinical outcome in cancer patients, and a positive correlation was observed between NCKAP1 expression and cancer-associated fibroblast infiltration (P < 0.05). Gene Ontology analysis revealed that NCKAP1 regulates the actin cytoskeleton and interacts with proteins such as CYFIP1, ABI2, WASF2, and BRK1. IHC staining showed significantly lower NCKAP1 levels in KIRC tissues compared to normal tissues. Overexpression of NCKAP1 in KIRC cell lines reduced cell proliferation, invasion, and migration (P < 0.05). NCKAP1 was also positively correlated with macrophage, neutrophil, and CD4+ T cell infiltration (P < 0.001). NCKAP1 may serve as a prognostic and immunological marker and may be a therapeutic target for KIRC.

Role of NCKAP1 in the Defective Phagocytic Function of Microglia-Like Cells Derived from Rapidly Progressing Sporadic ALS

Microglia plays a key role in determining the progression of amyotrophic lateral sclerosis (ALS), yet their precise role in ALS has not been identified in humans. This study aimed to identify a key factor related to the functional characteristics of microglia in rapidly progressing sporadic ALS patients using the induced microglia model, although it is not identical to brain resident microglia. After confirming that microglia-like cells (iMGs) induced by human monocytes could recapitulate the main signatures of brain microglia, step-by-step comparative studies were conducted to delineate functional differences using iMGs from patients with slowly progressive ALS [ALS(S), n = 14] versus rapidly progressive ALS [ALS(R), n = 15]. Despite an absence of significant differences in the expression of microglial homeostatic genes, ALS(R)-iMGs preferentially showed defective phagocytosis and an exaggerated pro-inflammatory response to LPS stimuli compared to ALS(S)-iMGs. Transcriptome analysis revealed that the perturbed phagocytosis seen in ALS(R)-iMGs was closely associated with decreased NCKAP1 (NCK-associated protein 1)-mediated abnormal actin polymerization. NCKAP1 overexpression was sufficient to rescue impaired phagocytosis in ALS(R)-iMGs. Post-hoc analysis indicated that decreased NCKAP1 expression in iMGs was correlated with the progression of ALS. Our data suggest that microglial NCKAP1 may be an alternative therapeutic target in rapidly progressive sporadic ALS.

NCK-associated protein 1 regulates metastasis and is a novel prognostic marker for colorectal cancer

Metastatic colorectal cancer (CRC) remains a substantial problem for mortality and requires screening and early detection efforts to increase survival. Epithelial-mesenchymal transition (EMT) and circulation of tumor cells in the blood play important roles in metastasis. To identify a novel target for metastasis of CRC, we conducted a gene microarray analysis using extracted RNA from the blood of preclinical models. We found that NCK-associated protein 1 (NCKAP1) was significantly increased in the blood RNA of patient-derived xenograft (PDX) models of colon cancer. In the NCKAP1 gene knockdown-induced human colon cancer cell lines HCT116 and HT29, there was a reduced wound healing area and significant inhibition of migration and invasion. As the result of marker screening for cytoskeleton and cellular interactions, CRC treated with siRNA of NCKAP1 exhibited significant induction of CDH1 and phalloidin expression, which indicates enhanced adherent cell junctions and cytoskeleton. In HCT116 cells with a mesenchymal state induced by TGFβ1, metastasis was inhibited by NCKAP1 gene knockdown through the inhibition of migration, and there was increased CTNNB1 expression and decreased FN expression. We established metastasis models for colon cancer to liver transition by intrasplenic injection shRNA of NCKAP1-transfected HCT116 cells or by implanting tumor tissue generated with the cells on cecal pouch. In metastasis xenograft models, tumor growth and liver metastasis were markedly reduced. Taken together, these data demonstrate that NCKAP1 is a novel gene regulating EMT that can contribute to developing a diagnostic marker for the progression of metastasis and new therapeutics for metastatic CRC treatment.

NCKAP1 is a Prognostic Biomarker for Inhibition of Cell Growth in Clear Cell Renal Cell Carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. Nck-associated protein 1 (NCKAP1) is associated with poor prognosis and tumor progression in several cancer types, but the function and prognostic value of NCKAP1 in ccRCC remain poorly understood. Methods: Using the Ualcan database, we evaluated the correlation between NCKAP1 expression and clinical features of ccRCC. These data were validated by immunohistochemical staining for NCKAP1 in a cohort of ccRCC patients. We assessed the prognostic value of NCKAP1 using GEPIA2 survival analysis. NCKAP1 function was characterized in vitro and in vivo using NCKAP1-overexpression ACHN cell lines. The LinkedOmics and GSCALite databases were used to investigate identify potential NCKAP1-targeted medicines that may play a role in the treatment of ccRCC. The impact of NCKAP1 expression on immune infiltration was also evaluated. Results: NCKAP1 was significantly downregulated in ccRCC and correlated with advanced clinicopathological features and poor prognosis. Overexpression of NCKAP1 in ACHN cells reduced proliferation, invasion and migration capacity in vitro and inhibited tumor growth in vivo. According to the LinkedOmics, GSCALite and TIMER databases, NCKAP1 and related genes function primarily in ribosomal signaling, oxidative phosphorylation, TGF-β, and EMT-related signaling pathways. NCKAP1 was also shown to positively correlate with immune cell types, biomarkers, and immune checkpoints in ccRCCs. Conclusions: NCKAP1 may play a vital tumor-suppressive role in ccRCC and is potentially a useful prognostic biomarker.

MiR-140-5p inhibits the proliferation, migration and invasion of vascular smooth muscle cells by suppressing the expression of NCKAP1.

The occurrence of aortic dissection is related to the proliferation and metastasis of vascular smooth muscle cells. In our present study, we found that the expression of miR-140-5p was inhibited in the wall of abdominal aorta of aortic dissection patients. However, the mechanism of miR-140-5p in the development of aortic dissection is unclear. We detected the expression of miR-140-5p and NCK Associated Protein 1 (NCKAP1) in blood vessel of aortic dissection patients and normal people by PCR. Next, we established the miR-140-5p overexpression and miR-140-5p inhibition vascular smooth muscle cells (CRL-1999 cells). The BrdU assays, wound healing assays and transwell assays were performed to detect the proliferation and invasion ability of these cells. Finally, luciferase reporter assay was performed to detect the relationship between miR-140-5p and NCKAP1. The expression of miR-140-5p was suppressed in blood vessel of aortic dissection patients, and the levels of NCKAP1 in those tissues were upregulated. Overexpression of miR-140-5p inhibited the proliferation, migration and invasion of vascular smooth muscle cells. miR-140-5p targeted and suppressed the expression of NCKAP1. miR-140-5p repressed the proliferation, migration and invasion of vascular smooth muscle cells by targeting and inhibiting the expression of NCKAP1. Furthermore, the results of our study suggest new strategies and targets for the clinical treatment of arterial dissection.

Redox proteomics reveals an interdependence of redox modification and location of adhesome proteins in NGF-treated PC12 cells

Proteomics studies have revealed that adhesomes are assembled from a plethora of proteins at integrin-mediated cellular contact sites with the extracellular matrix. By combining dimedone-trapping of sulfenylated proteins with the purification of the adhesome complex, we extended previous proteomics approaches on adhesomes to a redox proteomic analysis. This added a new aspect of adhesome complexity as individual adhesome proteins change their redox state in response to environmental signals. As model system, rat pheochromocytoma PC12 cells were studied in contact with type IV collagen and in response to nerve growth factor (NGF). NGF stimulates the endogenous production of reactive oxygen species (ROS) and the formation of neurite-like cell protrusions, which are anchored to the substratum via adhesomes. Dimedone detects the reversible oxidation of cysteine thiol groups into sulfenic acid groups which was used in proteomic analysis of adhesome proteins revealing that sulfenylation and location of proteins mutually influence each other. For some proteins, identified by the redox proteomics approach, among them Nck-associated protein-1 (Nap-1), proximity ligation analysis and co-immunoprecipitation assays proved that protein sulfenylation sites colocalize with adhesomes of protrusions. In conclusion, the suprastructural composition and function of adhesomes is redox-regulated by ROS. Of interest in this respect, isoform-selective pharmacological inhibition of NADPH-oxidases (Noxs) reduced the adhesomal location of the collagen-binding α1β1 integrin and the length of the outgrowing neurites, indicative of a role of Nox isoforms in the redox-regulation of adhesomes. Thus, our novel redox proteomics approach not only revealed redox-modifications and the potential redox-regulation of adhesomes and their constituents but it may also provide a tool to analyze the ROS-stimulated neurite repair of peripheral neurons.

NCKAP1 improves patient outcome and inhibits cell growth by enhancing Rb1/p53 activation in hepatocellular carcinoma

In our previous report, we identified miR-34c-3p as an independent factor contributing to the carcinogenesis of hepatocellular carcinoma (HCC) by targeting NCK Associated Protein 1 (NCKAP1). NCKAP1 has been known to promote the malignancy of cancer cells by disrupting the structural stability of WAS protein family member 1 (WASF1) and is correlated with poor prognosis of patients in several cancer types. Our results, however, show that NCKAP1 is correlated with a favorable outcome in HCC patients. The underlying mechanism of this contradictory phenomenon is unknown. The current study was designed to explore the mechanism of NCKAP1 in HCC. As a result, clinicopathological correlations and results from in vivo and in vitro models indicated that NCKAP1 was a tumor suppressor gene. Cell cycle analysis suggested that NCKAP1 inhibit cells from entering G2/M phase. Western blot analysis showed that WASF1 was barely expressed in HCC cell lines compared to that of breast cancer cell lines, which serve as positive controls. Furthermore, Rb1 and p53 expression was upregulated in cell lines overexpressing NCKAP1. Expression of several cell cycle regulating proteins also varied in the HCC cell lines. In conclusion, although previous studies have identified NCKAP1 as a cell invasion promoter by binding to WASF1, we found that NCKAP1 is a tumor suppress gene that modulates the cell cycle of HCC cell lines by targeting Rb1/p53 regulation.

A Nck‐associated protein 1‐like protein affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice

SummaryWater deficit is a major environmental threat affecting crop yields worldwide. In this study, a drought stress‐sensitive mutant drought sensitive 8 (ds8) was identified in rice (Oryza sativa L.). The DS8 gene was cloned using a map‐based approach. Further analysis revealed that DS8 encoded a Nck‐associated protein 1 (NAP1)‐like protein, a component of the SCAR/WAVE complex, which played a vital role in actin filament nucleation activity. The mutant exhibited changes in leaf cuticle development. Functional analysis revealed that the mutation of DS8 increased stomatal density and impaired stomatal closure activity. The distorted actin filaments in the mutant led to a defect in abscisic acid (ABA)‐mediated stomatal closure and increased ABA accumulation. All these resulted in excessive water loss in ds8 leaves. Notably, antisense transgenic lines also exhibited increased drought sensitivity, along with impaired stomatal closure and elevated ABA levels. These findings suggest that DS8 affects drought sensitivity by influencing actin filament activity.

NCK Associated Protein 1 Modulated by miRNA-214 Determines Vascular Smooth Muscle Cell Migration, Proliferation, and Neointima Hyperplasia.

BackgroundMicroRNA miR‐214 has been implicated in many biological cellular functions, but the impact of miR‐214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown.Methods and ResultsExpression of miR‐214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR‐214 in serum‐starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR‐214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)—a major component of the WAVE complex that regulates lamellipodia formation and cell motility—was negatively regulated by miR‐214 in VSMCs. Luciferase assays showed that miR‐214 substantially repressed wild‐type but not the miR‐214 binding site mutated version of NCKAP1 3′ untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR‐214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR‐214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR‐214–mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR‐214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury.ConclusionsWe uncovered an important role of miR‐214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR‐214 represents a potential therapeutic target for vascular diseases.

Fast neutron-induced structural rearrangements at a soybean NAP1 locus result in gnarled trichomes.

Key messageThree adjacent and distinct sequence rearrangements were identified at a NAP1 locus in a soybean mutant. Genetic dissection and validation revealed the function of this gene in soybean trichome development.A soybean (Glycine max (L.) Merr.) gnarled trichome mutant, exhibiting stunted trichomes compared to wild-type, was identified in a fast neutron mutant population. Genetic mapping using whole genome sequencing-based bulked segregant analysis identified a 26.6 megabase interval on chromosome 20 that co-segregated with the phenotype. Comparative genomic hybridization analysis of the mutant indicated that the chromosome 20 interval included a small structural variant within the coding region of a soybean ortholog (Glyma.20G019300) of Arabidopsis Nck-Associated Protein 1 (NAP1), a regulator of actin nucleation during trichome morphogenesis. Sequence analysis of the candidate allele revealed multiple rearrangements within the coding region, including two deletions (approximately 1–2 kb each), a translocation, and an inversion. Further analyses revealed that the mutant allele perfectly co-segregated with the phenotype, and a wild-type soybean NAP1 transgene functionally complemented an Arabidopsis nap1 mutant. In addition, mapping and exon sequencing of NAP1 in a spontaneous soybean gnarled trichome mutant (T31) identified a frame shift mutation resulting in a truncation of the coding region. These data indicate that the soybean NAP1 gene is essential for proper trichome development and show the utility of the soybean fast neutron population for forward genetic approaches for identifying genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-016-2735-x) contains supplementary material, which is available to authorized users.

Homologs of SCAR/WAVE complex components are required for epidermal cell morphogenesis in rice.

Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated: less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2 A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.

Global cellular responses to β-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin β-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 μM and 500 μM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro.

Lotus japonicus ARPC1 Is Required for Rhizobial Infection

Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.

Kette regulates actin dynamics and genetically interacts with Wave and Wasp.

During development of the Drosophila nervous system, kette is required for axonal growth and pathfinding. It encodes a highly conserved homolog of the Nck-associated protein 1 (NAP1) that genetically interacts with the Drosophila homolog of Nck, dock. We show that in vivo as well as in tissue culture models most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. The kette mutant phenotype can be suppressed by reducing the wave gene dose, demonstrating that kette antagonizes wave function. Overexpression of the wild-type Kette protein does not interfere with normal development, whereas expression of an activated, membrane-tethered Kette protein induces the formation of large F-actin bundles in both, tissue culture cells and in vivo. This gain-of-function phenotype is independent of wave but can be suppressed by reducing the wasp gene dose, indicating that Kette is able to regulate Wasp, to which it is linked via the Abelson interactor (Abi). Our data suggest a model where Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization.

Human Nck-associated protein1 and its binding protein affect the metabolism of beta-amyloid precursor protein with Swedish mutation

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system, and β-amyloid precursor protein (βAPP) plays a pivotal role in AD pathology. We previously reported that the suppression of human Nck-associated protein 1 (Nap1) whose expression was down-regulated in sporadic AD led to apoptosis in human neuroblastoma cells, and also its binding protein, hNap1BP was identified. Here, we examined whether these molecules were involved in the regulation of βAPP metabolism. Human Nap1 and hNap1BP were found not to effect the amount of intracellular βAPP but induced sAPPα secretion. Interestingly, they didn't reduce but slightly increased the extracellular level of Aβ. Furthermore, neither human Nap1 nor hNap1BP influenced the ratio of Aβ42/43 to total Aβ. Taken together, human Nap1 and hNap1BP may play a role in regulation of β-secretase activity in the processing of βAPP.