Coiled-coil Domain-containing Research Articles

Characterization of the novel tumor-suppressor gene CCDC67 in papillary thyroid carcinoma.

BackgroundSome studies showed an association of coiled-coil domain-containing (CCDC) genes with cancers. Our previous limited data specifically suggested a possible pathogenic role of CCDC67 in papillary thyroid cancer (PTC), but this has not been firmly established. The present study was to further investigate and establish this role of CCDC67 in PTC.ResultsThe expression of CCDC67, both at mRNA and protein levels, was sharply down-regulated in PTC compared with normal thyroid tissues. Lower CCDC67 expression was significantly associated with aggressive tumor behaviors, such as advanced tumor stages and lymph node metastasis, as well as BRAF mutation. Introduced expression of CCDC67 in TPC-1 cells robustly inhibited cell proliferation, colony formation and migration, induced G1 phase cell cycle arrest, and increased cell apoptosis.MethodsPrimary PTC tumors and matched normal thyroid tissues were obtained from 200 unselected patients at the initial surgery for detection of CCDC67 mRNA and protein by RT-PCR and Western blotting analyses, respectively. Genomic DNA sequencing was performed to detect BRAF mutation in PTC tumors. Clinicopathological data were retrospectively reviewed for correlation analyses. PTC cell line TPC-1 with stable transfection of CCDC67 was used to investigate the functions of CCDC67.ConclusionsThis large study demonstrates down-regulation of CCDC67 in PTC, an inverse relationship between CCDC67 expression and PTC aggressiveness and BRAF mutation, and a robust inhibitory effect of CCDC67 on PTC cellular activities. These results are consistent with CCDC67 being a novel and impaired tumor suppressor gene in PTC, providing important prognostic and therapeutic implications for this cancer.

Highlights of This Issue

CHCHD2 encodes a coiled-coil helix coiled-coil helix domain-containing protein that maps close to EGFR, and it is recurrently coamplified in non–small cell lung cancer (NSCLC), which accounts for more deaths per year than any other cancer. CHCHD2 protein is consistently upregulated in NSCLC, and its knockdown impairs cell proliferation, migration, and mitochondrial respiration. CHCHD2 is predominantly mitochondrial, and it is required for the stable assembly of oxidative phosphorylation components. The CHCHD2 interactome also includes an oncogenic transcription factor. Hence, CHCHD2 appears to function through mitochondrial and nuclear interactions, and, along with EGFR, appears to be a driver of 7p11.2 amplification and the cancer phenotype.Anticancer drugs targeting microtubule dynamics are widely prescribed for a broad range of cancers. However, drug resistance and severe side effects in treated patients limit their effectiveness. Lindström and colleagues show that citral dimethyl acetal (CDA), a citral analogue that specifically targets the nuclear activity of γ-tubulin, is a member of the tubulin family but has no effect on microtubule dynamics. CDA kills tumor cells with an impaired retinoblastoma (RB1) tumor suppressor signaling pathway without affecting healthy cells. CDA's in vivo antitumorigenic activity paves the way for the development of a novel broad-range, targeted anticancer therapy that causes fewer side effects.Transglutaminase-2 (TG2) is a vital cross-linking enzyme in the tumor microenvironment that has been linked to cancer progression, but its precise role appears to be context dependent. Cellura and colleagues found that TG2 inhibits invasion of colorectal cancer cells in vitro. Using a cell model of metastasis and analysis in sections taken from patients, a novel mechanism is described by which TG2 is downregulated in advanced colorectal cancer. Amplification of chromosome 13 leads to enhanced levels of miR-19 in metastatic cells, inhibiting TG2 to promote invasion. This study provides an important link between chromosomal instability, miRNA expression, and TG2 status, with functional effects on colorectal cancer cell behavior.The hypermethylated in cancer 1 (HIC1) gene encodes a sequence-specific transcriptional repressor that is frequently silenced in many solid tumors. Janeckova and colleagues used a conditional knock-out of Hic1 to identify genes regulated by this repressor. Hic1 ablation in primary fibroblasts and intestinal epithelia resulted in upregulation of Toll-like receptor 2 (TLR2), a critical mediator of prosurvival signal transduction pathways. In chemically induced colon cancer, Hic1 loss caused formation of highly proliferative Tlr2-positive neoplastic lesions. This study reveals that the tumor-suppressive function of Hic1 is related to its inhibitory action on signaling mediated by the Tlr2 receptor present on tumor cells.

The Coiled-Coil Domain Containing 80 (ccdc80) gene regulates gadd45β2 expression in the developing somites of zebrafish as a new player of the hedgehog pathway.

The Coiled-Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down-regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken, and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in-silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80-loss-of-function during embryonic development and verified its interaction with gadd45β2 in somitogenesis.

Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.

Targeting and Import Mechanism of Coiled-coil Helix Coiled-coil Helix Domain-containing Protein 3 (ChChd3) into the Mitochondrial Intermembrane Space

Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX(9)C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys(193), and the active site cysteine in Mia40, Cys(55). Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.

Loss of Coiled-Coil Domain Containing 80 Negatively Modulates Glucose Homeostasis in Diet-Induced Obese Mice

Coiled-coil domain containing 80 (Ccdc80) is a secreted protein highly enriched in mouse and human white adipose tissue (WAT) that plays an important role during adipocyte differentiation in vitro. To investigate the physiological function of Ccdc80 in energy and glucose homeostasis, we generated mice in which the gene encoding Ccdc80 was disrupted. Mice lacking Ccdc80 showed increased sensitivity to diet-induced hyperglycemia and glucose intolerance while displaying reduced glucose-stimulated insulin secretion in vivo. Gene expression analysis by microarray revealed that only 10 transcripts were simultaneously altered in pancreas, skeletal muscle, and WAT from Ccdc80(-/-) mice, including some components of the circadian clock. Expression of the core clock member Arntl/Bmal1 was reduced whereas that of the oscillating transcription factors Dbp and Tef was increased in all tissues examined. Furthermore, knockdown of Ccdc80 in 3T3-L1 cells led to an increase of Dbp mRNA levels during adipocyte differentiation, suggesting that Ccdc80 might be involved in the regulation of this gene in a cell-autonomous manner. Importantly, transcriptional alterations in Ccdc80(-/-) mice were associated with changes in feeding behavior, increased caloric intake, decreased energy expenditure, and obesity. Taken together, our results suggest that Ccdc80 is a novel modulator of glucose and energy homeostasis during diet-induced obesity.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Ccdc80-l1 Is Involved in Axon Pathfinding of Zebrafish Motoneurons

Axon pathfinding is a subfield of neural development by which neurons send out axons to reach the correct targets. In particular, motoneurons extend their axons toward skeletal muscles, leading to spontaneous motor activity. In this study, we identified the zebrafish Ccdc80 and Ccdc80-like1 (Ccdc80-l1) proteins in silico on the basis of their high aminoacidic sequence identity with the human CCDC80 (Coiled-Coil Domain Containing 80). We focused on ccdc80-l1 gene that is expressed in nervous and non-nervous tissues, in particular in territories correlated with axonal migration, such as adaxial cells and muscle pioneers. Loss of ccdc80-l1 in zebrafish embryos induced motility issues, although somitogenesis and myogenesis were not impaired. Our results strongly suggest that ccdc80-l1 is involved in axon guidance of primary and secondary motoneurons populations, but not in their proper formation. ccdc80-l1 has a differential role as regards the development of ventral and dorsal motoneurons, and this is consistent with the asymmetric distribution of the transcript. The axonal migration defects observed in ccdc80-l1 loss-of-function embryos are similar to the phenotype of several mutants with altered Hedgehog activity. Indeed, we reported that ccdc80-l1 expression is positively regulated by the Hedgehog pathway in adaxial cells and muscle pioneers. These findings strongly indicate ccdc80-l1 as a down-stream effector of the Hedgehog pathway.

Differential effects on p53-mediated cell cycle arrest vs. apoptosis by p90

p53 functions as a central node for organizing whether the cell responds to stress with apoptosis or cell cycle arrest; however, the molecular events that lead to apoptotic responses are not completely understood. Here, we identified p90 (also called Coiled-Coil Domain Containing 8) as a unique regulator for p53. p90 has no obvious effects on either the levels of p53 or p53-mediated cell cycle arrest but is specifically required for p53-mediated apoptosis upon DNA damage. Notably, p90 is crucial for Tip60-dependent p53 acetylation at Lys120, therefore facilitating activation of the proapoptotic targets. These studies indicate that p90 is a critical cofactor for p53-mediated apoptosis through promoting Tip60-mediated p53 acetylation.

A new bidirectional promoter from the human genome

Both human and other mammalian genomes contain a number of closely linked gene pairs transcribed in opposite directions. Bioinformatic analysis suggests that up to 10% of human genes are arranged in this way. This work reports cloning of a human genome fragment that separates two head-to-head oriented genes located at 2p13.1 and encoding hypothetical proteins with unknown functions: CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. The intergenic region CCDC142-TTC31 overlaps with a CpG island and contains a number of potential binding sites for transcription factors. This fragment functions as a bidirectional promoter in the system of luciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. Vectors containing oppositely oriented genes of two fluorescent proteins: green (EGFP) and red (DsRed2), separated by a fragment of the CCDC142-TTC31 intergenic region, were constructed. Transfection of HEK293 cells with these vectors resulted in simultaneous expression of both fluorescent proteins. The promoter activity was also determined for truncated versions of the intergenic region. The minimal promoter fragment contained Inr, BRE, and DPE elements characteristic for TATA-less promoters. Thus, a novel bidirectional promoter was cloned from the human genome; it can be used for simultaneous constitutive expression of two different genes in human cells.

ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.

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.

Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.