Coatomer Protein Research Articles

New Insights From Imputed Whole-Genome Sequence-Based Genome-Wide Association Analysis and Transcriptome Analysis: The Genetic Mechanisms Underlying Residual Feed Intake in Chickens.

Poultry feed constitutes the largest cost in poultry production, estimated to be up to 70% of the total cost. Moreover, there is pressure on the poultry industry to increase production to meet the protein demand of humans and simultaneously reduce emissions to protect the environment. Therefore, improving feed efficiency plays an important role to improve profits and the environmental footprint in broiler production. In this study, using imputed whole-genome sequencing data, genome-wide association analysis (GWAS) was performed to identify single-nucleotide polymorphisms (SNPs) and genes associated with residual feed intake (RFI) and its component traits. Furthermore, a transcriptomic analysis between the high-RFI and the low-RFI groups was performed to validate the candidate genes from GWAS. The results showed that the heritability estimates of average daily gain (ADG), average daily feed intake (ADFI), and RFI were 0.29 (0.004), 0.37 (0.005), and 0.38 (0.004), respectively. Using imputed sequence-based GWAS, we identified seven significant SNPs and five candidate genes [MTSS I-BAR domain containing 1, folliculin, COP9 signalosome subunit 3, 5′,3′-nucleotidase (mitochondrial), and gametocyte-specific factor 1] associated with RFI, 20 significant SNPs and one candidate gene (inositol polyphosphate multikinase) associated with ADG, and one significant SNP and one candidate gene (coatomer protein complex subunit alpha) associated with ADFI. After performing a transcriptomic analysis between the high-RFI and the low-RFI groups, both 38 up-regulated and 26 down-regulated genes were identified in the high-RFI group. Furthermore, integrating regional conditional GWAS and transcriptome analysis, ras-related dexamethasone induced 1 was the only overlapped gene associated with RFI, which also suggested that the region (GGA14: 4767015–4882318) is a new quantitative trait locus associated with RFI. In conclusion, using imputed sequence-based GWAS is an efficient method to identify significant SNPs and candidate genes in chicken. Our results provide valuable insights into the genetic mechanisms of RFI and its component traits, which would further improve the genetic gain of feed efficiency rapidly and cost-effectively in the context of marker-assisted breeding selection.

COPB2 promotes metastasis and inhibits apoptosis of lung adenocarcinoma cells through functioning as a target of miR-216a-3p

BackgroundLung adenocarcinoma is a non-small cell lung cancer with a high mortality. There is little published data on the role of coatomer protein complex subunit β (COPB2) in lung adenocarcinoma. The current study aimed to explore the effects of COPB2 on lung adenocarcinoma cells.MethodsThe differential expression of COPB2 in normal cells and lung adenocarcinoma cells was detected by quantitative real time-polymerase chain reaction (qRT-PCR) and Western blotting. Then, cell viability assay, flow cytometry and Transwell experiments were performed to study the effects of COPB2 on cell growth, apoptosis, migration and invasion. MiRNA targeting COPB2 was predicted by TargetScan and validated by luciferase assay, qRT-PCR and Western blotting. The effects of miRNA inhibitor on siCOPB2 were analyzed by rescue experiments. Finally, apoptosis and metastatic marker proteins were detected by Western blotting.ResultsCOPB2 was high-expressed in lung adenocarcinoma cells. Silencing COPB2 inhibited cell viability and cell metastasis, and significantly increased apoptosis. MiR-216a-3p was predicted to be able to target COBP2. Rescue experiment showed that miR-216a-3p inhibitor promoted cell viability, migration and invasion, and inhibited apoptosis of lung adenocarcinoma cells, partly reversed the effects of siCOPB2. Moreover, Western blotting showed that siCOPB2 up-regulated expressions of cleaved Caspase-3, Caspase-3, BCL2 associated X (Bax), and E-Cadherin, and down-regulated expressions of BCL2 apoptosis regulator (Bcl-2), N-Cadherin, and Vimentin, and the above effects were also partly reversed by miR-216a-3p inhibitor.ConclusionsHigh-expressed COPB2 promotes metastasis and inhibits apoptosis of lung adenocarcinoma cells through functioning as a target of miR-216a-3p.

Development of mite (Tetranychus urticae)-resistant transgenic Chinese cabbage using plant-mediated RNA interference

The two-spotted spider mite [Tetranychus urticae (Koch)] is a well-known omnivorous pest with approximately 3800 host crops, including Chinese cabbage (Brassica rapa ssp. pekinesis), one of the most important vegetable crops in Korea. We used plant-mediated RNA interference (RNAi) to produce mite-resistant Chinese cabbage lines and analyzed the molecular and phenotypic characteristics of the transgenic plants. A RNAi vector was constructed containing complementary fragments of the gene encoding coatomer protein complex subunit 2 (COPB2) derived from T. urticae, with both sense- and antisense-orientation, and the phosphinothricin acetyltransferase (Bar) gene, which was used as a selection marker. After Agrobacterium-mediated transformation, T1 transgenic lines, selected from 24 T0 transgenic lines, were generated by bud-pollination. The bioassay and reverse transcription quantitative PCR results indicated that the ‘CO 17-24’ T1 transgenic line showed high mite resistance with nearly a 100% mortality rate, and expressed approximately 160 times more COPB2 hairpin RNA than the other T1 transgenic lines. A phenotypic analysis showed no difference in the 26 examined characteristics between non-transgenic and T1 transgenic lines. We confirmed that the ‘CO 17-24’ T1 transgenic line contained one copy of the T-DNA inserted into the intergenic region between Bra039383 and Bra039384. And the inserted T-DNA of the ‘CO 17’ T0 transgenic line was stably inherited in ‘CO 17-24’ without any variation in the internal structure of the T-DNA. In conclusion, ‘CO 17-24’ is a candidate for an elite line in a breeding program to develop mite-resistant Chinese cabbage. This study demonstrated the effectiveness of the plant-mediated RNAi technique in developing mite-resistant Chinese cabbage, and it will provide useful information for developing pest-resistant plants.

COPZ1 depletion in thyroid tumor cells triggers type I IFN response and immunogenic cell death.

The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.

Upregulation of the Coatomer Protein Complex Subunit beta 2 (COPB2) Gene Targets microRNA-335-3p in NCI-H1975 Lung Adenocarcinoma Cells to Promote Cell Proliferation and Migration.

BackgroundThe coatomer protein complex subunit beta 2 (COPB2) gene is upregulated and promotes cell proliferation in some cancer cells. This study aimed to investigate the role of microRNA (miRNA) targeting by COPB2 gene expression in human lung adenocarcinoma cell lines, including NCI-H1975 cells.Material/MethodsCOPB2 expression in normal human bronchial epithelial cells and lung adenocarcinoma cells was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. NCI-H1975 human lung adenocarcinoma cells were transfected with short-interfering COPB2 (siCOPB2). Cell apoptosis and cell proliferation were evaluated by flow cytometry and Cell Counting Kit-8 (CCK-8) assays, respectively. The transwell assay evaluated cell migration. Targeting of miR-335-3p by COPB2 was predicted using TargetScan 7.2 and verified using a dual-luciferase reporter assay in NCI-H1975 cells. MiR-335-3p mimics were transfected into NCI-H1975 cells. The further functional analysis included detection of protein expression for cyclin D1, tissue inhibitor matrix metalloproteinase-1 (TIMP-1), matrix metallopeptidase 9 (MMP9), Bcl-2, and Bax, to verify the role of miR-335-3p targeting by COPB2 in lung adenocarcinoma cells.ResultsCOPB2 was upregulated in lung adenocarcinoma cells and was a direct target of miR-335-3p mimics. COPB2 knockdown promoted cell apoptosis, inhibited cell migration and proliferation in NCI-H1975 cells. The effects of COPB2 knockdown on NCI-H1975 cells were increased by miR-335-3p mimics, which also further reduced the expression levels of cyclin D1, MMP9, and Bcl-2 and further increased TIMP-1 and Bax by siCOPB2.ConclusionsThis study showed that COPB2 was the functional target of miR-335-3p in NCI-H1975 human adenocarcinoma cells.

Control of western corn rootworm via RNAi traits in maize: lethal and sublethal effects of Sec23 dsRNA.

RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

High expression of COPB2 predicts adverse outcomes: A potential therapeutic target for glioma.

AimsTo evaluate the clinical significance of coatomer protein complex subunit beta 2 (COPB2) in patients with glioma using a bioinformatics analysis.MethodsOncomine, GEO, and The Cancer Genome Atlas databases were used to examine the COPB2 transcript levels in glioma tissues. Gene expression profiles with clinical information from low‐grade glioma and glioblastoma (GBM) projects were analyzed for associations between COPB2 expression and clinicopathologic characteristics. Kaplan‐Meier survival and Cox regression analyses were used for survival analysis. Gene set enrichment analysis (GSEA) was conducted to screen the pathways involved in COPB2 expression. Gene set variation analysis (GSVA) and correlograms were performed to verify the correlations between COPB2 and inflammatory responses. Canonical correlation analyses examined whether COPB2‐high patients have more infiltrating inflammatory and immune cells.Results COPB2 was highly expressed in gliomas and high COPB2 expression correlated with shorter overall survival time and several poor clinical prognostic variables. GSEA indicated that some immune‐related pathways and other signaling pathways in cancer were associated with the COPB2‐high phenotype. The GSVA and canonical correlation analysis demonstrated that COPB2 expression was closely linked to inflammatory and immune responses, and higher immune cell infiltration.Conclusions COPB2 may be a potential prognostic biomarker and an immunotherapeutic target for glioma.

Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery

The western flower thrips, Frankliniella occidentalis, is a major pest that damages a wide variety of crops and vegetables. Following extensive use of insecticides, it has developed high levels of resistance to almost all groups of insecticides due to its high reproduction rate and short generation time. Therefore, an alternative pest control strategy, such as RNA interference (RNAi)-based control, is essential. To establish an ingestion RNAi-based control, a total of 57 genes involved in various biological processes were selected, and their double-stranded RNAs (dsRNA) were delivered to an insecticide-susceptible strain of F. occidentalis via the leaf disc-feeding method using a bioassay chamber optimized by 3D printing. The mortality of dsRNA-ingested thrips was examined every 24 h until 120 h post-treatment. Of the 57 genes screened, dsRNAs of the Toll-like receptor 6, apolipophorin, coatomer protein subunit epsilon and sorting and assembly machinery component were most lethal when ingested by thrips. The dsRNA-fed thrips showed substantially reduced transcription levels of target genes, demonstrating that the observed mortality was likely due to RNAi. When these genes were tested for ingestion RNAi against an insecticide-resistant strain of F. occidentalis, bioassay results were similar. In conclusion, this study provides the first evidence that ingestion RNAi can be lethal to F. occidentalis, a mesophyll sucking pest, and further suggests that transgenic plants expressing hairpin RNA of these essential genes can be employed to control insecticide-resistant thrips.

COPB2 is up‐regulated in breast cancer and plays a vital role in the metastasis via N‐cadherin and Vimentin

Breast cancer (BC) is a common malignant tumour for the adult female and its relative incidence has increased continuously in recent years. The primary molecular mechanisms of breast tumourigenesis remain unclear. With the sequencing technology, we found that coatomer protein complex subunit beta 2 (COPB2) gene is overexpressed in breast cancer tissues. However, the biological function of COPB2 in BC has yet to be determined. This current research demonstrates, significant up‐regulation of COPB2 in tissues of breast cancer while comparing the adjacent normal tissue both invalidated cohort and TCGA cohort. Up‐regulated expression of COPB2 was correlated with lymph node metastasis (LNM) and oestrogen receptor (ER) in the TCGA cohort and a high level of COPB2 was associated with age and lymph node metastasis in the validated cohort. Besides, logistic analysis illustrated in BC patient COPB2 expression, tumour size, age, ER and disease stage were independent high‐risk factors of LNM. Loss of function experiments revealed that down‐regulation of COPB2 could inhibit capacities of proliferation and cell invasion in MDA‐MB‐231 and BT‐549 cell lines. Moreover, underexpression of COPB2 could decrease the EMT‐related protein N‐cadherin and vimentin which may lead to cell invasion. This current research provides new shreds of evidence that COPB2 overexpression shows significant character in the progression of breast cancer. To best of our knowledge, our findings indicated that COPB2 was vital oncogene which was associated with breast cancer.

H2O2 induces nuclear transport of the receptor tyrosine kinase c-MET in breast cancer cells via a membrane-bound retrograde trafficking mechanism

Reactive oxygen species (ROS) are cellular by-products produced from metabolism and also anticancer agents, such as ionizing irradiation and chemotherapy drugs. The ROS H2O2 has high rates of production in cancer cells because of their rapid proliferation. ROS oxidize DNA, protein, and lipids, causing oxidative stress in cancer cells and making them vulnerable to other stresses. Therefore, cancer cell survival relies on maintaining ROS-induced stress at tolerable levels. Hepatocyte growth factor receptor (c-MET) is a receptor tyrosine kinase overexpressed in malignant cancer types, including breast cancer. Full-length c-MET triggers a signal transduction cascade from the plasma membrane that, through downstream signaling proteins, up-regulates cell proliferation and migration. Recently, c-MET was shown to interact and phosphorylate poly(ADP-ribose) polymerase 1 in the nucleus and to induce poly(ADP-ribose) polymerase inhibitor resistance. However, it remains unclear how c-MET moves from the cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of membrane-associated full-length c-MET into the nucleus of human MCF10A and MCF12A or primary breast cancer cells. We further show that knocking down either coatomer protein complex subunit γ1 (COPG1) or Sec61 translocon β subunit (SEC61β) attenuates the accumulation of full-length nuclear c-MET. However, a c-MET kinase inhibitor did not block nuclear c-MET transport. Moreover, nuclear c-MET interacted with KU proteins in breast cancer cells, suggesting a role of full-length nuclear c-MET in ROS-induced DNA damage repair. We conclude that a membrane-bound retrograde vesicle transport mechanism facilitates membrane-to-nucleus transport of c-MET in breast cancer cells.

Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

Silencing of COPB2 inhibits the proliferation of gastric cancer cells and induces apoptosis via suppression of the RTK signaling pathway.

Emerging studies have reported that coatomer protein complex subunit β2 (COPB2) is overexpressed in several types of malignant tumor; however, to the best of our knowledge, no studies regarding COPB2 in gastric cancer have been published thus far. Therefore, the present study aimed to determine the significance and function of COPB2 in gastric cancer. COPB2 expression in gastric cancer cell lines was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, lentivirus-short hairpin RNA (shRNA) COPB2 (Lv-shCOPB2) was generated and used to infect BGC-823 cells to analyze the effects of COPB2 on the cancerous phenotype. The effects of shRNA-mediated COPB2 knockdown on cell proliferation were detected using MTT, 5-bromo-2-deoxyuridine and colony formation assays. In addition, the effects of COPB2 knockdown on apoptosis were analyzed by flow cytometry. Nude mice and fluorescence imaging were used to characterize the regulation of tumor growth in vivo, and qPCR and immunohistochemistry were subsequently conducted to analyze COPB2 expression in xenograft tumor tissues. Furthermore, a receptor tyrosine kinase (RTK) signaling pathway antibody array was used to explore the relevant molecular mechanisms underlying the effects of COPB2 knockdown. The results revealed that COPB2 mRNA was abundantly overexpressed in gastric cancer cell lines, whereas knockdown of COPB2 significantly inhibited cell growth and colony formation ability, and led to increased cell apoptosis in vitro. The tumorigenicity assay revealed that knockdown of COPB2 reduced tumor growth in nude mice, and fluorescence imaging indicated that the total radiant efficiency of mice in the Lv-shCOPB2-infected group was markedly reduced compared with the mice in the Lv-shRNA control-infected group in vivo. The antibody array assay revealed that the levels of phosphorylation in 23 target RTKs were significantly reduced: In conclusion, COPB2 was highly expressed in gastric cancer cell lines, and knockdown suppressed colony formation and promoted cell apoptosis via inhibiting the RTK signaling and its downstream signaling cascade molecules. Therefore, COPB2 may present a valuable target for gene silencing strategy in gastric cancer.

Transcriptomic analysis of the Aquaporin (AQP) gene family interactome identifies a molecular panel of four prognostic markers in patients with pancreatic ductal adenocarcinoma

BackgroundThis study aimed to assess the differential gene expression of aquaporin (AQP) gene family interactome in pancreatic ductal adenocarcinoma (PDAC) using data mining techniques to identify novel candidate genes intervening in the pathogenicity of PDAC. MethodTranscriptome data mining techniques were used in order to construct the interactome of the AQP gene family and to determine which genes members are differentially expressed in PDAC as compared to controls. The same techniques were used in order to evaluate the potential prognostic role of the differentially expressed genes. ResultsTranscriptome microarray data of four GEO datasets were incorporated, including 142 primary tumor samples and 104 normal pancreatic tissue samples. Twenty differentially expressed genes were identified, of which nineteen were downregulated and one up-regulated. A molecular panel of four genes (Aquaporin 7 – AQP7; Archain 1 – ARCN1; Exocyst Complex Component 3 – EXOC3; Coatomer Protein Complex Subunit Epsilon – COPE) were identified as potential prognostic markers associated with overall survival. ConclusionThese outcomes should be further assessed in vitro in order to fully understand the role of these genes in the pathophysiological mechanism of PDAC.

A genome-wide analysis of coatomer protein (COP) subunits of apicomplexan parasites and their evolutionary relationships

BackgroundProtein secretion is an essential process in all eukaryotes including organisms belonging to the phylum Apicomplexa, which includes many intracellular parasites. The apicomplexan parasites possess a specialized collection of secretory organelles that release a number of proteins to facilitate the invasion of host cells and some of these proteins also participate in immune evasion. Like in other eukaryotes, these parasites possess a series of membrane-bound compartments, namely the endoplasmic reticulum (ER), the intermediate compartments (IC) or vesicular tubular clusters (VTS) and Golgi complex through which proteins pass in a sequential and vectorial fashion. Two sets of proteins; COPI and COPII are important for directing the sequential transfer of material between the ER and Golgi complex.ResultsHere, using in silico approaches, we identify the components of COPI and COPII complexes in the genome of apicomplexan organisms. The results showed that the COPI and COPII protein complexes are conserved in most apicomplexan genomes with few exceptions. Diversity among the components of COPI and COPII complexes in apicomplexan is either due to the absence of a subunit or due to the difference in the number of protein domains. For example, the COPI epsilon subunit and COPII sec13 subunit is absent in Babesia bovis, Theileria parva, and Theileria annulata genomes. Phylogenetic and domain analyses for all the proteins of COPI and COPII complexes was performed to predict their evolutionary relationship and functional significance.ConclusionsThe study thus provides insights into the apicomplexan COPI and COPII coating machinery, which is crucial for parasites secretory network needed for the invasion of host cells.

Association of CD40 SNPs with Moyamoya in a Chinese children population

Background: The etiology of Moyamoya disease (MMD) remains unknown to a large extent. Immune and inflammation dysfunction may play a role in the pathogenesis of this rare disease. Coexisting Kawasaki disease (KD) with MMD were reported and both diseases have a feature of vasculopathy, raising the hypothesis that there may be some common pathologic factors. We investigated single nucleotide polymorphisms (SNPs) previously identified in KD and performed a genetic analysis among Chinese pediatric patients with MMD.Results: We analyzed patients’ DNA for the SNPs in B lymphoid tyrosine kinase, CD40, and coatomer protein complex beta-2 subunit, which had been associated with KD by literatures. Genotyping was performed by sequencing the genetic regions containing the SNPs with customized primers. A total of 5 genotype polymorphisms were examined among 48 pediatric MMD cases and 50 healthy controls. The mean age of MMD children was 6.72 ± 3.63 years old, while 7.31 ± 3.79 in controls. We found two SNPs of CD40 were associated with MMD. Polymorphisms rs4813003 major allele CC and rs1535045 minor allele TT were significantly higher in MMD cases. The other SNPs showed no statistical difference between MMD cases and controls.Conclusions: Our findings provide evidence that there may be a relationship between MMD and auto-immune dysfunction. We hypothesize that these genetic features may lead to the pathogenesis within the vascular wall. Further study regarding whether CD40 can function as the personalized target of MMD should be investigated in future.

Dual RXR motifs regulate nerve growth factor-mediated intracellular retention of the delta opioid receptor.

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

Abstract 1908: Search for mediators of the lethal effect of COPZ1 depletion in thyroid tumor cells

Abstract We previously identified COPZ1 as an example of "non-oncogene" addiction for thyroid cancer cells, since its silencing drastically impairs the viability. Notably, the inhibition of COPZ1 has no effect on normal cells such as primary and normal immortalized thyrocytes. COPZ1 belongs to the coatomer protein complex I, involved in assembly of coated vesicles on Golgi membranes, the retrograde transport of proteins in the ER-Golgi secretory pathway, endosome maturation, autophagy and lipid homeostasis. We found that COPZ1 depletion leads to abortive autophagy, ER stress, unfolded protein response (UPR) and apoptosis. To better identify intracellular pathways that are activated upon COPZ1 silencing and that contribute to cell death, we performed a gene expression profiling of COPZ1-depleted 8505C cells (anaplastic thyroid tumor cells), 72h after siRNA transfection, by using Affymetrix Gene ST 2.0 array. Hierarchical clustering was applied on most variable genes in the entire dataset and specific gene expression patterns were identified in COPZ1-silenced cells compared to the control (non targeting siRNA-transfected cells), by means of SAM software (q-value 0). Functional annotation analyses by means of DAVID 6.8 bioinformatic tool and Gene Set Enrichment Analysis were performed. We found 321 genes specifically modulated in COPZ1-depleted 8505C cells; the main altered functional categories dealt with cell cycle regulation, chromosome organization and protein metabolism. Specifically, gene sets associated to the UPR, interferon signalling, vesicular transport and the translation machinery were found upregulated, whereas those related to DNA metabolism, telomere maintenance, cell cycle check points, cytoskeleton and cell adhesion, non-coding RNA and mRNA metabolism were found downregulated. Furthermore, we performed a proteomic profiling of 8505C cellular extracts (at 48h after COPZ1 depletion) by a nLC-ESI-MS/MS analysis. Bioinformatic analysis identified 271 down-regulated and 291 up-regulated proteins compared to the control. Overall, GeneTrail2 analysis revealed that the most changing pathways are related to RNA metabolism involved in splicesome and RNA processing, thus likely explaining the extensive transcriptional remodelling observed in the later time point. More interestingly, the gene expression profiling was also carried out in Nthy-ori 3-1 cells (immortalized normal human thyroid cells) that, unlike tumor cells, are resistant to cell death after COPZ1 depletion. We found no significant differentially expressed genes in COPZ1-depleted cells in comparison to the control, in keeping with the absence of a lethal effect, despite COPZ1 silencing. In conclusion, we identified novel functions of COPZ1 and further pathway interactions, in addition to that already known, that can be considered other critical nodes in the oncogenic network whose inhibition will result in cell collapse. Citation Format: Maria Chiara Anania, Katia Todoerti, Italia Bongarzone, Elena Cetti, Tiziana Di Marco, Antonino Neri, Angela Greco. Search for mediators of the lethal effect of COPZ1 depletion in thyroid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1908.

Xenin is a novel anorexigen in goldfish (Carassius auratus).

Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the copa mRNA encoding xenin in goldfish (Carassius auratus) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of copa cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that copa mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in copa mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in copa mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of copa mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish.

COPB2 suppresses cell proliferation and induces cell cycle arrest in human colon cancer by regulating cell cycle-related proteins.

Coat proteins (COPs), including the major types clathrin, COPI and COPII, play a considerable role in intracellular transport by initiating the formation of transport vesicles. Coatomer protein complex subunit β2 (COPB2) is one of the seven subunits that make up a COPI complex. In the present study, we found that COPB2 was highly expressed in human colon cancer specimens. However, to date, there have been no reports describing the functions of COPB2 in human colon cancer cells. In this study, we analyzed the functions of COPB2 in the proliferation and cell cycle arrest of human RKO and HCT116 colon cancer cells by using lentivirus-mediated RNAi infection. Our results demonstrated that the silencing of COPB2 in vitro could inhibit the proliferation and colony formation abilities of RKO and HCT116 cells. Furthermore, measurement of cell cycle distribution indicated that the downregulation of COPB2 could induce G0/G1 or S phase cell cycle arrest by regulating cell cycle-related proteins. In conclusion, our results suggest that COPB2 plays a key role in the proliferation and cell cycle progression of human RKO and HCT116 colon cancer cells, thus indicating that COPB2 might be a potential therapeutic target for the treatment of human colon cancer.

Identification and expression profile of an alpha-COPI homologous gene (COPA1) involved in high irradiance and salinity stress in Haematococcus pluvialis

In response to stresses, Haematococcus pluvialis accumulates triacylglycerols (TAGs) and astaxanthin, which are highly positively correlated in cells. Coatomer protein I (COPI) complexes play important roles in protein and lipid transportation between endoplasmic reticulum (ER) and Golgi apparatus. The proposed functions of COPI alpha-subunit (COPA1) were to maintain the integrity of endomembrane system and to regulate cellular physiological activities (such as autophagy, protein sorting, secretory trafficking and lipid metabolism). However, only scarce information has been reported on this subunit. In this study, the full-length cDNA encoding H. pluvialis COPA1 (HpCOPA1) and its promoter were cloned and further analyzed by bioinformatics tools. The predicted length of HpCOPA1 was 1234 amino acids (AAs), including WDR, Coatomer-WDAD and COPI-C motifs. The AA sequence of HpCOPA1 was highly identical (78%–99%) to other algal COPA1 and had a highly conserved WDR domain at the N terminus. The promoter of HpCOPA1 contained cis-elements in relation to light and salt stresses, such as AE-box, MNF1, CCAAT-box and TCA-element. Moreover, real-time qPCR analyses showed that the mRNA level of HpCOPA1 was strongly induced by treatments with either high irradiance, or high salinity (45mM sodium acetate) or in combination. HpCOPA1 revealed a delayed but higher peak expression level in treatments with high salinity (12.5-fold at 4h) compared with high irradiance (4.7-fold at 2h). To investigate its subcellular localization, HpCOPA1 was expressed in tobacco lower epidermal leaf cells in fusion with GFP. The results showed that HpCOPA1 was localized to plasma membrane and endomembrane system, partly similar to the localization of COPA1 in higher plants and animals. Taken together, the present results would contribute to the further elucidation of the regulatory mechanisms underlying the biosynthesis and transportation of lipids and astaxanthin in H. pluvialis.