C2 Domain-containing Research Articles

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells

High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Pathological Significance and Prognostic Role of WWC1 in Upper Urinary Tract Urothelial Cancer.

WW and C2 domain-containing 1 (WWC1) protein is a suppressor of malignancies. However, there is no information on the pathological significance of WWC1 in upper urinary tract cancer (UTUC). In this study, WWC1 immunoreactivity was investigated in 152 non-metastatic UTUC samples. The relationships between WWC1 expression and grade, pT stage, proliferative index (using an antibody to Ki-67), and the immunohistochemical expression of matrix metalloproteinase (MMP)-2 and -9 were evaluated. WWC1 expression was negatively associated with tumor grade and pT stage (p<0.001). Positive expression of WWC1 was a better predictor of the UTUC recurrence and subsequent metastasis, and the multivariate analysis showed that WWC1 expression was a significant predictor of subsequent metastasis (hazard ratio=0.29, p=0.020). WWC1 expression inversely correlated with the proliferative index (odds ratio=2.59, p=0.023) and expression of MMP9 (odds ratio=2.19, p=0.040) but not with MMP2 expression, by multivariate analyses. WWC1 expression was negatively associated with malignant aggressiveness via the suppression of cancer cell proliferation and MMP9 expression in patients with UTUC. This suggests WWC1 to be a useful predictor and novel therapeutic target in patients with UTUC.

Endometrial gene expression profiling of recurrent implantation failure after in vitro fertilization.

Recurrent implantation failure (RIF) is diagnosed when good-quality embryos repeatedly fail to implant after transfer in several in vitro fertilization (IVF) treatment cycles. Different expression profiles in maternal mRNAs could be referring to many diseases including RIF. This study aimed to reveal significantly dysregulated selected genes expression between healthy fertile women and RIF patients in the implantation window days of the natural menstrual cycle. MME, WWC1, TNC, and FOXP3 genes were chosen as target genes regarding their possible relations with the implantation process. Pathways with these genes were identified and the relationship between these pathways and RIF was investigated. In this study, the endometrial biopsy samples were collected in the secretory phase (cycle day 20-24) of the menstrual cycle from RIF patients (n = 34) and healthy fertile controls (n = 34). After "Pathway and network-oriented GWAS analysis" (PANOGA) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG) pathway analysis; "Membrane Metalloendopeptidase" (MME), "WW and C2 Domain Containing 1" (WWC1), "Tenascin C" (TNC) and "Forkhead Box P3" (FOXP3) genes were chosen as target genes by regarding their possible relation with implantation process. Detection of differences in mRNA expressions between the control group and RIF patients has been performed with the droplet digital PCR (ddPCR) method. Results of the study showed that MME and WWC1 genes expression levels are significantly (p < 0,05) up-regulated 4.9 and 5.2 times respectively and TNC gene expression level is significantly (p < 0,05) down-regulated 9 times in the RIF samples compared to the control group. However, no statistically significant difference was observed between the patient group and the control group in the expression of the FOXP3 gene (p < 0.05). Changes are observed in the expression of the renin-angiotensin system pathway in which the MME gene is involved in the implantation process. The increase in MME gene expression can be speculated to cause implantation failure by restricting the invasion of trophoblast cells. Increasing WWC1 gene expression in the Hippo signaling pathway inhibits "Yes-associated protein 1" (YAP) expression, which is a transcriptional cofactor. Inhibition of YAP protein expression may impair the implantation process by causing the failure of endometrial decidualization. The TNC gene is located in the focal adhesion pathway and this pathway reduces cell adhesion on the endometrial surface to facilitate the attachment of the embryo to the endometrium. The reason for implantation failure might be that the intercellular connections are not suitable for implantation as a result of decreased expression of the focal adhesion pathway in which the TNC gene is effective. Considering the relations between the pathways of the target genes and the implantation process, changes in the expression of target genes might be a cause of RIF.

Immune complexome analysis reveals the presence of immune complexes and identifies disease-specific immune complex antigens in saliva samples from patients with Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic autoimmune disease that mainly damages the salivary and lacrimal glands. Immune complex (IC) formation triggers local inflammation through IC deposition and decreased antigen function. Some ICs can leak from the lesion and into the saliva, but no salivary ICs have been reported to date. We used immune complexome analysis to comprehensively identify antigens incorporated into IC (IC-antigens) in saliva samples from patients with SS (n=9) or with xerostomia (n=7). Neutrophil defensin 1 (67%), small proline-rich protein 2D (67%), myeloperoxidase (44%), neutrophil elastase (44%), cathepsin G (33%), nuclear mitotic apparatus 1 (33%) and phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit gamma (33%) were identified as new IC-antigens specifically and frequently detected in the saliva of SS patients. Of these, neutrophil defensin 1, myeloperoxidase, neutrophil elastase and cathepsin G are neutrophil intracellular proteins, which suggests that repeated destruction of neutrophils due to abnormal autoimmunity may be involved in the pathogenesis of SS. We also analyzed serum samples from three SS patients. There was little overlap of IC-antigens between two of the samples (fewer than 30% of the IC-antigens in the saliva samples), suggesting that many ICs are formed locally and independently of the circulation. In addition, we found that four SS-specific salivary antigens show sequence homology with several proteins of oral microbiomes but no antigen has homology with Epstein-Barr virus proteins. The homology between some IC-antigens and oral microbiome proteins may indicate the impact of oral infection on local autoimmunity through molecular mimicry theory.

Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation.

Post-mitotic neurons do exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanged. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, the occurrence of de novo DNA methylation and its impacts have been much less investigated. In the present study, we showed that neuronal activation induces de novo DNA methylation at enhancer regions, which can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 domain-containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in synaptogenesis. We found that both DNMT1 and DNMT3a were required for neuronal activity-induced de novo DNA methylation of the Wwc1 enhancer. Taken together, we concluded that neuronal activity-induced de novo DNA methylation that affects gene expression has an impact on neuronal physiology that is comparable to that of DNA demethylation. Since the different requirements of DNMTs for germ cell and embryonic development are known, our findings also have considerable implications for future studies on epigenomics in the field of reproductive biology.

De Novo Coding Variants Are Strongly Associated with Tourette Syndrome

Based on the contribution of de novo coding variants to other neurodevelopmental disorders, and their utility for hypothesis-naive gene discovery, we whole-exome sequenced 325 Tourette syndrome trios (311 passed quality control) from the TIC Genetics consortium, and identified de novo sequence variants. We observed a strong association for de novo likely gene disrupting (LGD; insertion of premature stop codon, disruption of canonical splice site, or frameshift insertion/deletion) variants as well as de novo damaging variants (LGD + probably damaging missense [Mis3]) variants. We replicated these findings in 186 TS trios (173 after quality control) from the TSAICG Consortium, and performed a combined analysis strongly associating de novo LGD (RR 2.32, 95% CI 1.37-3.93, p = 0.002) and damaging (RR 1.37, 95% CI 1.11-1.69, p = 0.003) variants with TS risk. We estimate that de novo damaging variants in approximately 12% of patients contribute risk through the perturbation of approximately 400 risk genes. Finally, we leveraged recurrent de novo damaging variants and the TADA algorithm to identify one high confidence (false discovery rate (FDR) < 0.10) TS risk gene: WWC1 (WW and C2 Domain Containing 1); and three probable (FDR < 0.30) TS risk genes: CELSR3 (Cadherin EGF LAG Seven-Pass G-Type Receptor 3); NIPBL (Nipped-B-Like); and FN1 (Fibronectin 1). These findings establish the contribution of de novo variants to TS risk and highlight that de novo based gene discovery is a viable path forward for this disorder.

WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway.

Our data define an essential in vivo function for WWC proteins as regulators of canonical and noncanonical Hippo signaling in hepatic cell growth and liver tumorigenesis. Thus, expression of WWC proteins may serve as novel prognostic factors in human liver carcinoma. (Hepatology 2018;67:1546-1559).

WWC3 regulates the Wnt and Hippo pathways via Dishevelled proteins and large tumour suppressor 1, to suppress lung cancer invasion and metastasis.

The scaffolding protein WWC (WW and C2-domain containing) family is known to regulate cell proliferation and organ size via the Hippo signalling pathway. However, the expression level of WWC3 in human tumours and the mechanisms underlying its role in cellular signal transduction have not yet been reported. Herein, we explored the potential roles of WWC3 in lung cancer cells and the corresponding molecular mechanisms. We found low WWC3 expression in both lung cancer cell lines and lung cancer specimens, which was associated with low differentiation, advanced pTNM stage, positive lymph node metastasis, and poor prognosis in patients with lung cancer. Moreover, the overexpression of WWC3 inhibited the proliferation and invasiveness of lung cancer cells. These effects were mediated by the inhibition and stimulation of the Wnt and Hippo pathways, respectively, in vitro and in vivo. Specifically, WWC3 interacts with Dishevelled (Dvl) proteins, prevents casein kinase 1ϵ from phosphorylating Dvls, and inhibits β-catenin nuclear translocation to inhibit the Wnt pathway. Deleting the WW and C-terminal PDZ-binding domains of WWC3 abrogated these effects. Moreover, the interaction of WWC3 with Dvls reduced the interaction between WWC3 and large tumour suppressor 1 (LATS1), as well as decreasing LATS1 phosphorylation to increase the nuclear importation of yes-associated protein (YAP) and attenuate the Hippo pathway. Deleting the WW domain of WWC3 abrogated this effect. These findings demonstrate the molecular interplay between WWC3, Dvls, and LATS1, and reveal a link between the Wnt and Hippo pathways, which provides a potential target for clinical intervention in lung cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Randomly organized lipids and marginally stable proteins: A coupling of weak interactions to optimize membrane signaling

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6–5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Screening for differentially expressed genes between left- and right-sided colon carcinoma by microarray analysis

Left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC) differ in their genetic susceptibilities to neoplastic transformation. The present study identified 11 genes that were differentially expressed in LSCC and RSCC by expression profiling with microarray analysis. Compared with RSCC, the human genes for L-lactate dehydrogenase B chain (LDHB), cyclin-dependent kinase 4 inhibitor D (CDKN2D), phosphatidylinositol-4-phosphate-3-kinase C2 domain-containing subunit α (PI3KC2α), protocadherin fat 1 (FAT; a human protein that closely resembles the Drosophila tumor suppressor, fat) and dual specificity protein phosphatase 2 (DUSP2) were upregulated in LSCC. By contrast, genes for ubiquitin D (UBD), casein kinase-1 binding protein (CK1BP), synaptotagmin-13 (SYT1), zinc finger protein 560 (ZNF560), pleckstrin homology domain-containing family B member 2 (PLEKHB2) and IgGFc-binding protein (FCGBP) were downregulated in LSCC compared with RSCC. A quantitative polymerase chain reaction (qPCR) analysis revealed that the mRNA levels of UBD and CK1BP in LSCC were significantly lower compared with those in RSCC (P=0.033 and P= 0.005, respectively), whereas the mRNA levels of LDHB and CDKN2D in LSCC were significantly higher compared with those in RSCC (P=0.008 and P=0.017, respectively). Western blot and immunohistochemical analyses demonstrated that the expression of CDKN2D in LSCC was significantly higher compared with that in RSCC, while the expression of UBD in LSCC was significantly lower compared with that in RSCC. The present study provides important insights into the understanding of the molecular genetic basis for the different biological behaviors observed between LSCC and RSCC. These insights may therefore serve as a basis for the identification of novel colon cancer markers and therapeutic targets.

The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.

Pollen Proteins Bind to the C-terminal Domain of Nicotiana alata Pistil Arabinogalactan Proteins

Pollen tube growth is influenced by interaction between pollen proteins and the pistil extracellular matrix. The transmitting tract-specific glycoprotein (NaTTS) and 120-kDa glycoprotein (120K) are two pistil arabinogalactan proteins (AGPs) that share a conserved C-terminal domain (CTD) and directly influence pollen tubes in Nicotiana alata. 120K and other extracellular matrix proteins are taken up and transported to vacuoles of growing pollen tubes. We hypothesize that signaling and trafficking processes inside pollen tubes are important for controlling pollen tube growth. We performed a yeast two-hybrid screen of pollen cDNAs using sequences from 120K and NaTTS as baits. We found that an S-RNase-binding protein (SBP1), a C2 domain-containing protein (NaPCCP), and a putative cysteine protease bound to the AGP baits. SBP1 from Petunia hybrida and Solanum chacoense is a putative E3 ubiquitin ligase that binds to S-RNase and other proteins. C2 domain-containing proteins bind lipids and can regulate myriad cellular processes. Cysteine proteases are often associated with the degradation of vacuolar proteins. Expression analysis revealed that transcripts for these proteins are expressed in mature pollen. NaPCCP and NaSBP1 were characterized further because of their potential roles in signaling and trafficking. In vitro pull-down assays verified binding between maltose-binding protein (MBP) fusions, MBP::NaPCCP or MBP::NaSBP1 and glutathione S-transferase (GST), GST::AGP CTD fusions. NaSBP1 binds to the AGP CTDs through its helical and RING domains. NaPCCP binds through its C-terminal region. Binding between NaPCCP and NaSBP1 and the pistil AGPs may contribute to signaling and trafficking inside pollen tubes growing in planta.

CC2D2A, Encoding A Coiled-Coil and C2 Domain Protein, Causes Autosomal-Recessive Mental Retardation with Retinitis Pigmentosa

Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca(2+)-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR.

Ege A, a Novel C2 Domain Containing Protein, Is Essential for GPCR-Mediated Gene Expression in Dictyostelium

During early stages of development, expression of aggregative genes in Dictyostelium is regulated by G protein-linked signaling pathways. We have isolated an aggregation-deficient mutant from a restriction enzyme-mediated insertional mutagenesis screen and have obtained its cDNA. Since the mutant expresses prestarvation genes but fails to express early genes, such as cAR1 and GP80, during development, we designated it early gene expression A ( ege A). Ege A, encoding a cytosolic protein of 26 kDa, along with Ege B, belongs to a novel C2 domain-containing gene family. While Ege A mRNA is expressed during the first 2 h of development, Ege B is expressed at later stages. Ege A is not directly required for either G protein-mediated actin polymerization or activation of adenylyl cyclase. Ege A overexpressing and ege A − cells display similar phenotypes, suggesting that an optimal level of Ege A is required for proper function. Constitutive expression of a fully functional cAR1-YFP enables ege A − cells to form loose aggregates, but cAR1-YFP/ ege A − cells are still unable to express GP80, suggesting that losses of gene expression were not solely due to a lack of cAR1. Overexpression of PKAcat, the constitutively active subunit of PKA, does not rescue the ege A − phenotype, suggesting that PKA is not located downstream from Ege A in the signaling pathway. We propose that Ege A is a novel cytosolic component required by early gene expression.

Identification and cDNA Cloning of a Novel Mammalian C2 Domain-Containing Phosphoinositide 3-Kinase, HsC2-PI3K

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signal transduction, cell transformation and intracellular protein trafficking. Reverse-transcription polymerase chain reaction methods, using degenerate primers derived from the lipid kinase consensus region, were utilised to identify PI 3-kinases in the normal human breast. Here we report the cDNA cloning of a novel human PI 3-kinase isoform, HsC2-PI3K. This PI 3-kinase is most closely related to the recently described C2 domain-containing family of PI 3-kinases which includesDrosophilaPI3K_68D/cpk and murine cpk-m/p170. Sequence analysis suggests that HsC2-PI3K is a second distinct mammalian member of the C2 domain-containing PI 3-kinase family. Northern blot analysis of human tissues indicates that HsC2-PI3K is widely expressed. Fluorescencein situhybridisation has mapped HsC2-PI3K to chromosome 1q32.