Mutational screening through comprehensive bioinformatics analysis to detect novel germline mutations in the APC gene in patients with familial adenomatous polyposis (FAP).

BACKGROUND: Familial adenomatous polyposis (FAP) as a colon cancer predisposition syndrome is an autosomal-dominant inherited condition and is diagnosed by the progress of hundreds or thousands of adenomatous colonic polyps in the colon. This study aims at the nature and effect of Adenomatous Polyposis Coli (APC) gene mutations in FAP tumorigenesis. METHODS: The genetic screening of 59 FAP Iranian patients in 10 families was performed by polymerase chain reactions and the direct sequencing of the entire coding exons of the APC gene. To do linkage haplotype analysis and multiplex PCR-based microsatellite examination, six short tandem repeat loci were selected in this gene. To evaluate and predict the potentially deleterious effects, comprehensive bioinformatics pathogenicity assays were used. RESULTS: A total of 12 germline heterozygous and homozygous nucleotide variations were identified. They included two missense mutations, four nonsense mutations, which would lead to the truncated and nonfunctional protein products, four synonymous or silent variations, and two nucleotide deletions of 1 to 5 bp or frameshift mutations. In addition, three novel heterozygous nonsense mutations were found in exons 10, 14, and 15 of the gene. There was also p.Arg653Met as a novel heterozygote mutation in exon 14 of the gene. CONCLUSIONS: Bioinformatics analysis and three-dimensional structural modeling predicted that these missense and nonsense mutations generally are associated with the deleted or truncated domains of APC and have functional importance and mainly affected the APC protein. These findings may provide evidence for the progress of potential biomarkers and help to understand the role of the APC gene in FAP.

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors.

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.

The 15-Amino Acid Repeat Region of Adenomatous Polyposis Coli Is Intrinsically Disordered and Retains Conformational Flexibility upon Binding ß-Catenin.

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via ß-catenin destruction. This destruction is mediated by ß-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC. More than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein that retains the ∼200-amino acid long' 15-amino acid repeat region'. This study demonstrates that the 15-amino acid repeat region of APC is intrinsically disordered. We investigated the backbone dynamics in the presence of ß-catenin and predicted residues that may contribute to transient secondary features. This study reveals that the 15-amino acid region of APC retains flexibility upon binding ß-catenin and that APC does not have a single, observable "highest-affinity" binding site for ß-catenin. This flexibility potentially allows ß-catenin to be more readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing.

Discovery of novel pyrazoline derivatives containing methyl-1H-indole moiety as potential inhibitors for blocking APC-Asef interactions.

A series of novel pyrazoline derivatives containing methyl-1H-indole moiety were discovered as potential inhibitors for blocking APC-Asef interactions. The top hit Q19 suggested potency of inhibiting APC-Asef interactions and attractive preference for human-sourced colorectal cells. It was already comparable with the previous representative and the positive control Regorafenib before further pharmacokinetic optimization. The introduction of methyl-1H-indole moiety realized the Mitochondrial affection thus might connect the impact on the protein-interaction level with the apoptosis events. The molecular docking simulation inferred that bringing trifluoromethyl groups seemed a promising approach for causing more key interactions such as H-bonds. This work raised referable information for further discovery of inhibitors for blocking APC-Asef interactions.

Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents.

There are very few generic methods to assess the stability and functional properties of membrane proteins solubilized in detergent. For this purpose, a thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can be used. An unfolding profile is obtained when the fluorochrome becomes fluorescent on reaction with cysteine residues that have been exposed during thermal denaturation of the protein population. The method was initially developed to optimize the stability of membrane proteins for crystallization studies, but in the course of our work we found many other applications. First, the assay can be used to study the binding of inhibitors, substrates, lipids, and other effectors to membrane proteins. Second, the assay can be used to understand the dynamics of proteins, allowing states to be defined by changes in accessibility of cysteine residues or by changes in specific amino acid interactions. Finally, the assay can be used to study state-dependent domain interactions, for example, as part of regulatory mechanisms. The CPM thermostability assay represents a broadly applicable and versatile tool for a wide range of applications in the functional and structural analysis of membrane proteins.

The binding affinity of PTPN13's tandem PDZ2/3 domain is allosterically modulated.

BACKGROUND: Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, is a large multi-domain non-transmembrane scaffolding protein with a molecular mass of 270 kDa. It is involved in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement. The modular structure of PTPN13 consists of an N-terminal KIND domain, a FERM domain, and five PDZ domains, followed by a C-terminal protein tyrosine phosphatase domain. PDZ domains are among the most abundant protein modules and they play a crucial role in signal transduction of protein networks. RESULTS: Here, we have analysed the binding characteristics of the isolated PDZ domains 2 and 3 from PTPN13 and compared them to the tandem domain PDZ2/3, which interacts with 12 C-terminal residues of the tumour suppressor protein of APC, using heteronuclear multidimensional NMR spectroscopy. Furthermore, we could show for the first time that PRK2 is a weak binding partner of PDZ2 and we demonstrate that the presence of PDZ3 alters the binding affinity of PDZ2 for APC, suggesting an allosteric effect and thereby modulating the binding characteristics of PDZ2. A HADDOCK-based molecular model of the PDZ2/3 tandem domain from PTPN13 supports these results. CONCLUSIONS: Our study of tandem PDZ2/3 in complex with APC suggests that the interaction of PDZ3 with PDZ2 induces an allosteric modulation within PDZ2 emanating from the back of the domain to the ligand binding site. Thus, the modified binding preference of PDZ2 for APC could be explained by an allosteric effect and provides further evidence for the pivotal function of PDZ2 in the PDZ123 domain triplet within PTPN13.

Design, synthesis and biological evaluation of 2-H pyrazole derivatives containing morpholine moieties as highly potent small molecule inhibitors of APC-Asef interaction.

Mutated adenomatous polyposis coli (APC) selectively combining with Asef has been reported to be implicated in promoting colon cancer proliferation, invasion and metastasis in several cancer biotherapy studies. However, there were universally resistance and harsh terms in disrupting APC-Asef interaction in biotherapy. Under the circumstances small-molecule inhibitors as the new APC interface could resolve the problems. In this research, a series of novel dihydropyrazole derivatives containing morpholine as high potent interaction inhibitors between APC and Asef were first synthesized after selection by means of docking simulation and virtual screening. Afterwards they were evaluated interaction inhibition of APC-Asef and pharmacological efficiency both in vitro and in vivo utilizing orthotopic transplantation model with multi-angle of view. Among them, compound 7g exhibited most excellent anti-proliferation activities against HCT116 cells with IC50 of 0.10 ±â€¯0.01 µM than Regorafenib (IC50 = 0.16 ±â€¯0.04 µM). The results favored our rational design intention and provides a new class of small-molecule inhibitors available for the development of colon tumor therapeutics targeting APC-Asef interaction inhibitions.

A sensitive fluorometric DNA nanobiosensor based on a new fluorophore for tumor suppressor gene detection.

In this study, a sensitive fluorescent DNA nanobiosensor has been developed to determine DNA sequence of a well-known tumor suppressor gene, Adenomatous Polyposis Coli (APC). The design of the nanobiosensor was carried out using a synthetic organic ligand as a new fluorophore. The response mechanism of the nanobiosensor was based on DNA hybridization. The new fluorophore was assembled on gold nanoparticles (Au NPs) to enhance the sensitivity of the nanobiosensor response. The fabricated DNA nanobiosensor showed a fluorescence emission at 477 nm by exciting wavelength of 360 nm. By addition of the ssDNA target, the fluorescent emission of the nanobiosensor enhanced linearly in the range from 3.3 × 10-10 to 1.1 × 10-9 mol L-1 with detection limit of 1.3 × 10-11 mol L-1. The proposed DNA nanobiosensor responded selectively to its complementary strand in comparison with non-complementary and three mismatched bases. The nanobiosensor had also a fast response time with acceptable repeatability. Finally, the performance of the DNA nanobiosensor in biological fluid, serum plasma, was investigated and a satisfactory results were obtained.

A novel APC mutation identified in a large Chinese family with familial adenomatous polyposis and a brief literature review.

Familial adenomatous polyposis (FAP), an autosomal dominant disease, is a colon cancer predisposition syndrome that manifests as a large number of adenomatous polyps. Mutations in the Adenomatous polyposis coli (APC) gene are responsible for the majority of cases of FAP. The purpose of the present study was to report the clinical features of a Chinese family with FAP and screen for novel mutations using the targeted next­generation sequencing technology. Among the 29 family members, 12 were diagnosed of FAP. Based on an established filtering strategy and data analyses, along with confirmation by Sanger sequencing and co­segregation, a novel frameshift mutation c.1317delA (p.Ala440LeufsTer14) in exon 10 of the APC gene was identified. To the best of our knowledge, this mutation has not been reported prior to the present study. In addition, it was correlated with extra­colonic phenotypes featuring duodenal polyposis and sebaceous cysts in this family. This novel frameshift mutation causing FAP not only expands the germline mutation spectrum of the APC gene in the Chinese population, but it also increases the understanding of the phenotypic and genotypic correlations of FAP, and may potentially lead to improved genetic counseling and specific treatment for families with FAP in the future.

Truncated Adenomatous Polyposis Coli Mutation Induces Asef-Activated Golgi Fragmentation.

Adenomatous polyposis coli (APC) is a key molecule to maintain cellular homeostasis in colonic epithelium by regulating cell-cell adhesion, cell polarity, and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (Asef). The APC-activated Asef stimulates the small GTPase, which leads to decreased cell-cell adherence and cell polarity, and enhanced cell migration. In colorectal cancers, while truncated APC constitutively activates Asef and promotes cancer initiation and progression, regulation of Asef by full-length APC is still unclear. Here, we report the autoinhibition mechanism of full-length APC. We found that the armadillo repeats in full-length APC interact with the APC residues 1362 to 1540 (APC-2,3 repeats), and this interaction competes off and inhibits Asef. Deletion of APC-2,3 repeats permits Asef interactions leading to downstream signaling events, including the induction of Golgi fragmentation through the activation of the Asef-ROCK-MLC2. Truncated APC also disrupts protein trafficking and cholesterol homeostasis by inhibition of SREBP2 activity in a Golgi fragmentation-dependent manner. Our study thus uncovers the autoinhibition mechanism of full-length APC and a novel gain of function of truncated APC in regulating Golgi structure, as well as cholesterol homeostasis, which provides a potential target for pharmaceutical intervention against colon cancers.

Identification and characterization of functional single nucleotide polymorphisms (SNPs) in Axin 1 gene: a molecular dynamics approach.

Wnt signaling pathway has been reported to play crucial role in intestinal crypt formation and deregulation of this pathway is responsible for colorectal cancer initiation and progression. Axin 1, a scaffold protein, play pivotal role in the regulation of Wnt/ß-catenin signaling pathway and has been found to be mutated in several cancers; primarily in colon cancer. Considering its crucial role, a structural and functional analysis of missense mutations in Axin 1 gene was performed in this study. Initially, one hundred non-synonymous single nucleotide polymorphisms in the coding regions of Axin 1 gene were selected for in silico analysis. Six variants (G820S, G856S, E830K, L811V, L847V, and R767C) were predicted to be deleterious by combinatorial prediction. Further investigation of structural attributes confirmed two highly deleterious single nucleotide polymorphisms (G820S and G856S). Molecular dynamics simulation demonstrated variation in different structural attributes between native and two highly deleterious Axin 1 mutant models. Finally, docking analysis showed variation in binding affinity of mutant Axin 1 proteins with two destruction complex members, GSK3ß and adenomatous polyposis. The results collectively showed the deleterious effect of the above predicted single nucleotide polymorphisms on the Axin 1 protein structure and could prove to be an adjunct in the disease genotype-phenotype correlation studies.

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots.

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10- 10 to 1.0 × 10- 6M with detection limit of 6.2 × 10- 11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.

Computational Framework for Prediction of Peptide Sequences That May Mediate Multiple Protein Interactions in Cancer-Associated Hub Proteins.

A considerable proportion of protein-protein interactions (PPIs) in the cell are estimated to be mediated by very short peptide segments that approximately conform to specific sequence patterns known as linear motifs (LMs), often present in the disordered regions in the eukaryotic proteins. These peptides have been found to interact with low affinity and are able bind to multiple interactors, thus playing an important role in the PPI networks involving date hubs. In this work, PPI data and de novo motif identification based method (MEME) were used to identify such peptides in three cancer-associated hub proteins-MYC, APC and MDM2. The peptides corresponding to the significant LMs identified for each hub protein were aligned, the overlapping regions across these peptides being termed as overlapping linear peptides (OLPs). These OLPs were thus predicted to be responsible for multiple PPIs of the corresponding hub proteins and a scoring system was developed to rank them. We predicted six OLPs in MYC and five OLPs in MDM2 that scored higher than OLP predictions from randomly generated protein sets. Two OLP sequences from the C-terminal of MYC were predicted to bind with FBXW7, component of an E3 ubiquitin-protein ligase complex involved in proteasomal degradation of MYC. Similarly, we identified peptides in the C-terminal of MDM2 interacting with FKBP3, which has a specific role in auto-ubiquitinylation of MDM2. The peptide sequences predicted in MYC and MDM2 look promising for designing orthosteric inhibitors against possible disease-associated PPIs. Since these OLPs can interact with other proteins as well, these inhibitors should be specific to the targeted interactor to prevent undesired side-effects. This computational framework has been designed to predict and rank the peptide regions that may mediate multiple PPIs and can be applied to other disease-associated date hub proteins for prediction of novel therapeutic targets of small molecule PPI modulators.

Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase.

Wnt/ß-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator ß-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.

3DBIONOTES: A unified, enriched and interactive view of macromolecular information.

With the advent of high throughput techniques like Next Generation Sequencing, the amount of biological information for genes and proteins is growing faster than ever. Structural information is also rapidly growing, especially in the cryo Electron Microscopy area. However, in many cases, the proteomic and genomic data are spread in multiple databases and with no simple connection to structural information. In this work we present a new web platform that integrates EMDB/PDB structures and UniProt sequences with different sources of protein annotations. The application provides an interactive interface linking sequence and structure, including EM maps, presenting the different sources of information at sequence and structural level. The web application is available at http://3dbionotes.cnb.csic.es.

APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane.

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.

Thr160 of Axin1 is critical for the formation and function of the ß-catenin destruction complex.

Upon binding of a Wnt ligand to the frizzled (FZD)-low density lipoprotein receptor related protein 5/6 (LRP5/6) receptor complex, the ß-catenin destruction complex, composed of Axin1, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), is immediately inactivated, which causes ß-catenin stabilization. However, the molecular mechanism of signal transduction from the receptor complex to the ß-catenin destruction complex is controversial. Here we show that Wnt3a treatment promotes the dissociation of the Axin1-APC complex in glioblastoma cells cultured in serum-free medium. Experiments with the GSK3 inhibitor BIO suggest that Axin1-APC dissociation was controlled by phosphorylation. Introduction of a phosphomimetic mutation into Thr160 of Axin1, located in the APC-binding region RGS, abrogated the interaction of Axin1 with APC. Consistent with these observations, the Axin1 phosphomimetic mutant lost the ability to reduce ß-catenin stability and to repress ß-catenin/TCF-dependent transcription. Taken together, our results suggest a novel mechanism of Wnt signaling through the dissociation of the ß-catenin destruction complex by Axin1 Thr160 modification.

APC promoter 1B deletion in seven American families with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation-dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele-specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42-98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States.

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLß and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3ß, and CK1 that targets ß-catenin/Armadillo (ß-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both ß-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates ß-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in ß-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.