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Background: Ferroptosis, a type of autophagy-dependent cell death, has been implicated in the pathogenesis of lung adenocarcinoma (LUAD). This study aimed to investigate the involvement of coatomer protein complex I subunit zeta 1 (COPZ1) in ferroptosis and ferritinophagy in LUAD., Methods: Publicly available human LUAD sample data were obtained from the TCGA database to analyze the association of COPZ1 expression with LUAD grade and patient survival. Clinical samples of LUAD and para-carcinoma tissues were collected. COPZ1-deficient LUAD cell model and xenograft model were established. These models were analyzed to evaluate tumor growth, lipid peroxidation levels, mitochondrial structure, autophagy activation, and iron metabolism., Results: High expression of COPZ1 was indicative of malignancy and poor overall survival. Clinical LUAD tissues showed increased COPZ1 expression and decreased nuclear receptor coactivator 4 (NCOA4) expression. COPZ1 knockdown inhibited xenograft tumor growth and induced apoptosis. COPZ1 knockdown elevated the levels of ROS, Fe 2+ and lipid peroxidation. COPZ1 knockdown also caused mitochondrial shrinkage. Liproxstatin-1, deferoxamine, and z-VAD-FMK reversed the effects of COPZ1 knockdown on LUAD cell proliferation and ferroptosis. Furthermore, COPZ1 was directly bound to NCOA4. COPZ1 knockdown restricted FTH1 expression and promoted NCOA4 and LC3 expression. NCOA4 knockdown reversed the regulation of iron metabolism, lipid peroxidation, and mitochondrial structure induced by COPZ1 knockdown. COPZ1 knockdown induced the translocation of ferritin to lysosomes for degradation, whereas NCOA4 knockdown disrupted this process., Conclusion: This study provides novel evidence that COPZ1 regulates NCOA4-mediated ferritinophagy and ferroptosis. These findings provide new insights into the pathogenesis and potential treatment of LUAD., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coat protein I (COPI) and Coat protein II (COPII) coated vesicles mediate protein transport in the early secretory pathway. Although several components of COPII vesicles have been shown to have an essential role in Arabidopsis gametogenesis, the function of COPI components in gametogenesis has not been studied in detail. COPI consists of a heptameric complex made of α, β, β', γ, δ, ɛ, and ζ-COP subunits and most subunits have several isoforms in Arabidopsis. We have found that two isoforms of the β'-COP subunit, β'1-COP and β'2-COP, are required for female and male gametophyte development. Reciprocal crosses between wild type plants and plants heterozygous for T-DNA insertions in β'1-COP and β'2-COP showed that β'1β'2-cop gametophytes are not transmitted., (© 2023. The Author(s).)

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

K2P channels, also known as two-pore domain K + channels, play a crucial role in maintaining the cell membrane potential and contributing to potassium homeostasis due to their leaky nature. The TREK, or tandem of pore domains in a weak inward rectifying K + channel (TWIK)-related K + channel, subfamily within the K2P family consists of mechanical channels regulated by various stimuli and binding proteins. Although TREK1 and TREK2 within the TREK subfamily share many similarities, β-COP, which was previously known to bind to TREK1, exhibits a distinct binding pattern to other members of the TREK subfamily, including TREK2 and the TRAAK (TWIK-related acid-arachidonic activated K + channel). In contrast to TREK1, β-COP binds to the C-terminus of TREK2 and reduces its cell surface expression but does not bind to TRAAK. Furthermore, β-COP cannot bind to TREK2 mutants with deletions or point mutations in the C-terminus and does not affect the surface expression of these TREK2 mutants. These results emphasize the unique role of β-COP in regulating the surface expression of the TREK family.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how β'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both β'-COP propeller domains are required to bind Glo3. An acidic patch on β'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or β'-COP abrogate the interaction in vitro, and loss of the β'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the β'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where β'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex., (© 2023 Xie et al.)

Mature astrocytes are characterized by a K + conductance (passive conductance) that changes with a constant slope with voltage, which is involved in K + homeostasis in the brain. Recently, we reported that the tandem of pore domains in a weak inward rectifying K + channel (TWIK1 or KCNK1) and TWIK-related K + channel 1 (TREK1 or KCNK2) form heterodimeric channels that mediate passive conductance in astrocytes. However, little is known about the binding proteins that regulate the function of the TWIK1/TREK1 heterodimeric channels. Here, we found that β-coat protein (COP) regulated the surface expression and activity of the TWIK1/TREK1 heterodimeric channels in astrocytes. β-COP binds directly to TREK1 but not TWIK1 in a heterologous expression system. However, β-COP also interacts with the TWIK1/TREK1 heterodimeric channel in a TREK1 dependent manner and enhances the surface expression of the heterodimeric channel in astrocytes. Consequently, it regulates TWIK1/TREK1 heterodimeric channel-mediated passive conductance in astrocytes in the mouse brain. Taken together, these results suggest that β-COP is a potential regulator of astrocytic passive conductance in the brain.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae . The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of β'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization., Competing Interests: SD, PX, NH, ND, JB, BX, JD, ES, LJ, JM, TG No competing interests declared, (© 2022, Date et al.)

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the highly aggressive malignancy types of head and neck squamous cell carcinomas; genes involved in the development of LSCC still need exploration., Methods: We downloaded expression profiles of 96 (85 in advanced stage and 11 in early stage) LSCC patients from TCGA-HNSC. Function enrichment and protein-protein interactions of genes in significant modules were conducted. Univariate and multivariate Cox regression analyses were performed to explore potential prognostic biomarkers for LSCC. The expression levels of genes at different stages were compared and visualized via boxplots. Immune infiltration was examined by the CIBERSORTx web-based tool and depicted with ggplot2. Gene set enrichment analysis (GSEA) was utilized to analyze functional enrichment terms and pathways. Immunohistochemical staining (IHC) was used to verify the expression of genes in the LSCC samples., Results: We identified 25 modules, including 3 modules significantly related to tumor stages of LSCC via weighted gene co-expression network analysis (WGCNA). UIMC1, NPM1, and DCTN4 in the module 'cyan', TARS in the module 'darkorange', and COPB2 and RYK in the module 'lightyellow' showed statistically significant relation to overall survival. The expression of COPB2, DCTN4, RYK, TARS, and UIMC1 indicated association with the change of fraction of immune cells in LSCC patients; two genes, COPB2 and RYK, indicated different expression in various tumor stages of LSCC. Finally, COPB2 and RYK showed high-expression in tumor tissues of advanced LSCC patients., Conclusions: Our study provided a potential perceptive in analyzing progression of LSCC cells and exploring prognostic genes., (© 2022. The Author(s).)

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A (BFA) resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA- and GCA (golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1 which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. IMPORTANCE Classical swine fever (CSF), a highly contact-infectious disease caused by classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection are still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

Coat protein I (COPI) is necessary for intra-Golgi transport and retrograde transport from the Golgi apparatus back to the endoplasmic reticulum. The key component of the COPI coat is the coatomer complex, which is composed of seven subunits (α/β/β'/γ/δ/ε/ζ) and is recruited en bloc from the cytosol onto Golgi membranes. In mammals and yeast, α- and β'-COP WD40 domains mediate cargo-selective interactions with dilysine motifs present in canonical cargoes of COPI vesicles. In contrast to mammals and yeast, three isoforms of β'-COP (β'1-3-COP) have been identified in Arabidopsis . To understand the role of Arabidopsis β'-COP isoforms in plant biology, we have identified and characterized loss-of-function mutants of the three isoforms, and double mutants were also generated. We have found that the trafficking of a canonical dilysine cargo (the p24 family protein p24δ5) is affected in β'-COP double mutants. By western blot analysis, it is also shown that protein levels of α-COP are reduced in the β'-COP double mutants. Although none of the single mutants showed an obvious growth defect, double mutants showed different growth phenotypes. The double mutant analysis suggests that, under standard growth conditions, β'1-COP can compensate for the loss of both β'2-COP and β'3-COP and may have a prominent role during seedling development.

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking., (© 2022. The Author(s).)

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC., Competing Interests: Declaration of interests I.S., P.S. and J.S. (in conjunction with the University of Toronto) are listed as inventors on a patent (publication number 20190091205) for the use of EMI1 (and structurally related analogues), midostaurin, gilteritinib and AZD7762 (and structurally related analogues) in the treatment of mutant EGFR-mediated non-small-cell lung cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: The aim of the present study was to investigate the contribution of Nav1.8 expressing corneal afferent neurons to the presence of ongoing pain in lacrimal gland excision (LGE)-induced dry eye., Methods: The proton pump archaerhodopsin-3/eGFP (ArchT/eGFP) was conditionally expressed in corneal afferents using Nav1.8-cre mice. Dry eye was produced by unilateral LGE. Real time place preference was assessed using a three-chamber apparatus. A neutral, unlit center chamber was flanked by one illuminated with a control light and one illuminated with an ArchT activating light. For real-time preference, animals were placed in the neutral chamber and tracked over five 10-minute sessions, with the lights turned on during the second and fourth sessions. In other studies, movement was tracked over three 10-minute sessions (the lights turned on only during the second session), with animals tested once per day over the course of 4 days. A local anesthetic was used to examine the role of ongoing corneal afferent activity in producing place preference., Results: The corneal afferent nerves and trigeminal ganglion cell bodies showed a robust eGFP signal in Nav1.8-cre;ArchT/eGFP mice. After LGE, Nav1.8-cre;ArchT/eGFP mice demonstrated a preference for the ArchT activating light paired chamber. Preference was prevented with pre-application to the cornea of a local anesthetic. Nav1.8-cre;ArchT/eGFP mice with sham surgery and LGE wild-type control mice did not develop preference., Conclusions: Results indicate LGE-induced persistent, ongoing pain, driven by Nav1.8 expressing corneal afferents. Inhibition of these neurons represents a potential strategy for treating ongoing dry eye-induced pain.

Several studies have suggested that coatomer protein complex subunit beta 2 (COPB2) may act as an oncogene in various cancer types. However, no systematic pan-cancer analysis has been performed to date. Therefore, the present study analyzed the potential oncogenic role of COPB2 using TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) datasets. The majority of the cancer types overexpressed the COPB2 protein, and its expression significantly correlated with tumor prognosis. In certain tumors, such as those found in breast and ovarian tissues, phosphorylated S859 exhibited high expression. It was found that mutations of the COPB2 protein in kidney and endometrial cancers exhibited a significant impact on patient prognosis. It is interesting to note that COPB2 expression correlated with the number of cancer-associated fibroblasts in certain tumors, such as cervical and endocervical cancers and colon adenocarcinomas. In addition, COPB2 was involved in the transport of substances and correlated with chemotherapy sensitivity. This is considered the first pan-tumor study, which provided a relatively comprehensive understanding of the mechanism by which COPB2 promotes cancer growth., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Biao Wu et al.)

Porcine Parvovirus (PPV) is a pathogen causing porcine reproductive disorders. Non-structural protein NS1 appears diverse functions acting as a predominant regulator in promoting PPV replication. In this study, we identified a PPV NS1 binding protein coatomer subunit epsilon (COPƐ), and found that COPƐ is a critical regulator during PPV replication. In NS1 transfected or PPV infected cells, COPƐ was interacted with NS1 and translocated into nucleus together with NS1. Knockout of COPƐ could inhibit PPV production by increasing the expression levels of IFN-β, while overexpression of COPƐ enhanced PPV production by reducing the expression levels of IFN-β. Furthermore, the domain mapping assay showed that the N-terminal amino acids domain of NS1 (25-EAFSYVF-31) were required for the interaction of COPƐ with NS1. Sequence alignment result displays that parvovirus NS1 (EAFSYVF) amino acids domain is highly conservative among PPV, CPV, FPV and MEV, and down-regulation of COPƐ could also significantly reduce the replication of these viruses. Notably, we found that the interaction of COPƐ with NS1 play an important role in promoting the production of type I interferon during PPV or CPV infection, which affect the replication of these viruses. Taken together, the results presented here show a novel function of NS1 interaction with COPƐ that regulates the parvovirus replication through modulating the type I interferons signaling pathway, provided a potential target for the control of parvovirus-associated diseases., (Copyright © 2021. Published by Elsevier B.V.)

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei., Competing Interests: The authors have declared that no competing interests exist.

Purpose: This study is aimed at investigating the expression, underlying biological function, and clinical significance of coatomer protein complex subunit beta 2 (COPB2) in hepatocellular carcinoma (HCC)., Methods: HCC-related data were extracted from The Cancer Genome Atlas (TCGA) database, International Cancer Genome Consortium (ICGC) database, and Gene Expression Omnibus (GEO) database. A logistic regression module was applied to analyze the relationship between the expression of COPB2 and clinicopathologic characteristics. The Cox proportional hazard regression model and Kaplan-Meier method were used for survival analysis. Gene set enrichment analysis (GSEA) was used to annotate the underlying biological functions. Loss-of-function experiments were conducted to determine the underlying mechanisms., Results: COPB2 was overexpressed in HCC, and high expression of COPB2 was significantly correlated with higher alpha fetoprotein (AFP) (odds ratio (OR) = 1.616, >20 vs. ≤20, p < 0.05), stage (OR = 1.744, III vs. I, p < 0.05), and grade (OR = 1.746, G4+G3 vs. G2+G1, p < 0.05). Kaplan-Meier survival analysis showed that HCC patients with high COPB2 expression had a worse prognosis than those with low COPB2 expression ( p < 0.0001 for TCGA cohort, p < 0.05 for ICGC cohort). The univariate Cox (hazard ratio (HR) = 1.068, p < 0.0001) and multivariate Cox (HR = 2.011, p < 0.05) regression analyses suggested that COPB2 was an independent risk factor. GSEA showed that mTOR and other tumor-related signaling pathways were differentially enriched in the high COPB2 expression phenotype. Silencing of COPB2 inhibited the proliferation, migration, and invasion abilities by suppressing epithelial-mesenchymal transition and mTOR signaling., Conclusion: COPB2 is a novel prognostic biomarker and a promising therapeutic target for HCC., Competing Interests: The authors report no conflicts of interest in this work., (Copyright © 2021 Jiayao Zhang et al.)

A 25-year-old woman with a history of juvenile idiopathic arthritis and rheumatoid factor-positive polyarthritis developed dyspnoea. Progressive cystic lung disease was diagnosed. Biomarkers of autoimmunity, such as antinuclear antibodies, antiextractable nuclear antigen antibodies, anti-SCL-70, rheumatoid factor, cyclic citrullinated peptide antibodies, c-antineutrophil cytoplasmic antibody and MPO, were found. No familial disease was reported. Despite lack of kidney manifestations, coatomer-associated protein subunit alpha syndrome was suggested. Type 1 interferon signature score was 40.8 (range, <2.3). A class 4 heterozygous mutation (c.725T>G, p.Val242Gly) was confirmed. Due to abnormal trafficking between the Golgi complex and the endoplasmic reticulum, a Mendelian monogenic autosomal dominant syndrome associating inflammatory arthritis with interstitial lung disease, with several high-titre autoantibodies, was identified. Treatment with tyrosine kinase inhibitors, Janus kinases-signal transducers and activators of transduction, may be beneficial., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Pathogenic COPA variants cause a Mendelian syndrome of immune dysregulation with elevated type I interferon signaling. COPA is a subunit of coat protein complex I (COPI) that mediates Golgi to ER transport. Missense mutations of the COPA WD40 domain impair binding and sorting of proteins targeted for ER retrieval, but how this causes disease remains unknown. Given the importance of COPA in Golgi-ER transport, we speculated that type I interferon signaling in COPA syndrome involves missorting of STING. We show that a defect in COPI transport causes ligand-independent activation of STING. Furthermore, SURF4 is an adapter molecule that facilitates COPA-mediated retrieval of STING at the Golgi. Activated STING stimulates type I interferon-driven inflammation in CopaE241K/+ mice that is rescued in STING-deficient animals. Our results demonstrate that COPA maintains immune homeostasis by regulating STING transport at the Golgi. In addition, activated STING contributes to immune dysregulation in COPA syndrome and may be a new molecular target in treating the disease., Competing Interests: Disclosures: T. Martinu reported grants from Sanofi and non-financial support from APCBio outside the submitted work. No other disclosures were reported., (© 2020 Deng et al.)

Heterozygous missense mutations in coatomer protein subunit α, COPA, cause a syndrome overlapping clinically with type I IFN-mediated disease due to gain-of-function in STING, a key adaptor of IFN signaling. Recently, increased levels of IFN-stimulated genes (ISGs) were described in COPA syndrome. However, the link between COPA mutations and IFN signaling is unknown. We observed elevated levels of ISGs and IFN-α in blood of symptomatic COPA patients. In vitro, both overexpression of mutant COPA and silencing of COPA induced STING-dependent IFN signaling. We detected an interaction between COPA and STING, and mutant COPA was associated with an accumulation of ER-resident STING at the Golgi. Given the known role of the coatomer protein complex I, we speculate that loss of COPA function leads to enhanced type I IFN signaling due to a failure of Golgi-to-ER STING retrieval. These data highlight the importance of the ER-Golgi axis in the control of autoinflammation and inform therapeutic strategies in COPA syndrome., Competing Interests: Disclosures: M. Wislez reported personal fees from Boeringher Ingelheim, Roche, MSD, BMS, Astra Zeneca, and Amgen outside the submitted work. Y.J. Crow reported "other" from Biogen outside the submitted work. No other disclosures were reported., (© 2020 Lepelley et al.)

Arginine methylation is a common posttranslational modification that modulates protein function. SCY1-like pseudokinase 1 (SCYL1) is crucial for neuronal functions and interacts with γ 2 -COP to form coat protein complex I (COPI) vesicles that regulate Golgi morphology. However, the molecular mechanism by which SCYL1 is regulated remains unclear. Here, we report that the γ 2 -COP-binding site of SCYL1 is arginine-methylated by protein arginine methyltransferase 1 (PRMT1) and that SCYL1 arginine methylation is important for the interaction of SCYL1 with γ 2 -COP. PRMT1 was colocalized with SCYL1 in the Golgi fraction. Inhibition of PRMT1 suppressed axon outgrowth and dendrite complexity via abnormal Golgi morphology. Knockdown of SCYL1 by small interfering RNA (siRNA) inhibited axon outgrowth, and the inhibitory effect was rescued by siRNA-resistant SCYL1, but not SCYL1 mutant, in which the arginine methylation site was replaced. Thus, PRMT1 regulates Golgi morphogenesis via SCYL1 arginine methylation. We propose that SCYL1 arginine methylation by PRMT1 contributes to axon and dendrite morphogenesis in neurons.

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Hausrath et al.)

SAG is an essential RING component of the Cullin-RING ligase (CRL) E3 ubiquitin ligase complex, which regulates diverse signaling pathways and biological processes, including cell apoptosis, embryonic development, angiogenesis, and tumorigenesis. In the present study, we revealed that SAG gene expression is upregulated in breast cancer cells and that SAG overexpression is associated with significant poorer survival in breast cancer, especially the luminal A subtype. We also detected highly correlated co-overexpression of SAG and COPB2 in breast cancers. Subsequent in vitro experiments demonstrated that SAG and COPB2 act cooperatively to stimulate breast cancer cell proliferation, migration and invasion. Our findings suggest that levels of SAG and COPB2 expression may be useful prognostic indicators in breast cancers and that SAG may be involved in COPB2-related signaling during breast cancer development.

Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome-level evidence of genome variations or RNA editing. In this work, the products of adenosine-to-inosine RNA editing in human and murine brain proteomes are identified using publicly available brain proteome LC-MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false-positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes are identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites have been previously reported using orthogonal methods, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors, CYFIP2, coatomer alpha. Also, differential editing between neurons and microglia is demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, the results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report that expression of the α-COP protein rescues disease phenotype in a severe mouse model of Spinal Muscular Atrophy (SMA). Lentiviral particles expressing α-COP were injected directly into the testes of genetically pure mouse strain of interest resulting in infection of the spermatagonial stem cells. α-COP was stably expressed in brain, skeletal muscle, and spinal cord without altering SMN protein levels. SMA mice transgenic for α-COP live significantly longer than their non-transgenic littermates, and showed increased body mass and normal muscle morphology at postnatal day 15. We previously reported that binding between SMN and α-COP is required for restoration of neurite outgrowth in cells lacking SMN, and we report similar finding here. Lentiviral-mediated transgenic expression of SMN where the dilysine domain in exon 2b was mutated was not able to rescue the SMA phenotype despite robust expression of the mutant SMN protein in brain, muscle and spinal cord. These results demonstrate that α-COP is a validated modifier of SMA disease phenotype in a mammalian, vertebrate model and is a potential target for development of future SMN-independent therapeutic interventions., (Published by Elsevier Inc.)

Reactive oxygen species (ROS) are cellular by-products produced from metabolism and also anticancer agents, such as ionizing irradiation and chemotherapy drugs. The ROS H 2 O 2 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 H 2 O 2 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., (© 2019 Chen et al.)

H5N1 influenza A virus (IAV) causes seasonal epidemics that represent a worldwide threat to public health. IAV relies on host factors for viral replication. PB1 N40 is translated from the fifth starting code (AUG) of PB1 mRNA, which is the product of the ribosomal scan omission. Here, we report the interactome landscape of H5N1 IAV PB1 N40 protein in chicken cells. The interacting complexes were captured by co-immunoprecipitation and analyzed by mass spectrometry. We identified 135 proteins as PB1 N40-interacting proteins. GO and Pathway analysis showed that proteins with biological functions such as protein localization and viral transcription and proteins related to signaling pathways of DNA replication and cell cycle were significantly enriched in virus-host interactions, suggesting the potential roles of them in infection with H5N1 IAV. Comparative analysis among H1N1 and H5N1 revealed conservation of the virus-host protein interaction between different subtypes or strains of influenza virus. ARCN1 was identified as a host interacting factor of H5N1 IAV PB1 N40 protein, which is the component of the coatomer. Knockdown of ARCN1 significantly decreased the titer of H5N1 IAV in chicken cells. BIOLOGICAL SIGNIFICANCE: Influenza A virus (IAV) is a great threat to public health and avian production. However, the manner in which avian IAV recruits the host cellular machinery for replication and how the host antagonizes the IAV infection was previously poorly understood. Here we present the viral-host interactome of the H5N1 IAV PB1 N40 protein and reveal its involvement with dozens of important host genes during the course of IAV infection., (Copyright © 2019. Published by Elsevier B.V.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We report here the finding of abnormal Golgi apparatus morphology in motor neuron like cells depleted of SMN as well as Golgi apparatus morphology in SMA patient fibroblasts. Rescue experiments demonstrate that this abnormality is dependent on SMN, but can also be rescued by expression of the COPI coatomer subunit alpha-COP. A motor neuron-like cell line containing an inducible alpha-COP shRNA was created to generate a parallel system to study knockdown of SMN or alpha-COP. Multiple assays of COPI-dependent intracellular trafficking in cells depleted of SMN demonstrate that alpha-COP function is suboptimal, including failed sequestration of plasma membrane proteins, altered binding of mRNA, and defective targeting and transport of Golgi-resident proteins., (Published by Elsevier B.V.)

Cytotoxic morula cells (MCs) and phagocytes are the circulating immunocytes of the colonial ascidian Botryllus schlosseri: Both these cells can synthesise amyloid fibrils, supporting the idea that physiological amyloidogenesis is involved in inflammation and modulation of immune responses. Intriguingly, amyloid of B. schlosseri immunocytes is made of two different proteins. MCs, the first cells to sense non-self and involved in the allorejection reaction between contacting genetically incompatible colonies, use melanin encapsulation as the principal method to fight non-self. They release amyloid fibrils formed by p102 protein that allow the packaging and deposit of melanin and other toxic molecules nearby the invader or in the contact region of incompatible colonies. Phagocytes release amyloid-based extracellular traps when challenged with microbes: their amyloid fibrils harbour BsAPP, an orthologue of the vertebrate amyloidogeneic protein APP. This strategy of immune response, present also in human neutrophils, allows phagocytes to block and engulf bacteria and fungi., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection. IMPORTANCE Human cytomegalovirus infection is a significant cause of disease in immunocompromised populations, individuals with heart disease, and recipients of solid organ and bone marrow transplants. HCMV is also the leading cause of infectious congenital birth defects. The majority of antivirals in clinical use target components of the virus to specifically inhibit replication. However, a major drawback of this approach is the emergence of resistance. An alternative approach is to target host factors that the virus requires for successful infection. In this study, multiple host factors were identified that were found to be essential for the production of newly infectious human cytomegalovirus. Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate and how cells function and provides potential targets for novel antivirals., (Copyright © 2018 McCormick et al.)

Objective: The present study aimed to explore the contribution of COPB2 (coatomer subunit beta) towards the tumorigenesis of cholangiocellular carcinomas and to elucidate the underlying mechanism(s)., Materials and Methods: Expression of COPB2 mRNA by RBE and QBC939 cholangiocellular carcinoma cell lines was determined by qRT-PCR. We, then, silenced COPB2 expression in RBE cells by infection with a COPB2-siRNA lentivirus and measured the proliferation, cell-cycle distribution, and apoptosis of transduced cells., Results: COPB2 was highly expressed in RBE and QBC939 cholangiocellular carcinoma cell lines. Infection with COPB2-siRNA lentivirus in RBE cells significantly decreased COPB2 expression. More so, silencing of COPB2 by COPB2-siRNA significantly suppressed the proliferation and promoted the apoptosis of RBE cells by arresting transduced cells in the G1 phase., Conclusions: Our results demonstrate that the COPB2 gene is highly expressed in cholangiocellular carcinoma cell lines, wherein knockdown inhibited the proliferation and promoted the arrest of cell-cycle progression and the apoptosis of cholangiocellular carcinomas. COPB2 may constitute an attractive target for therapeutic strategies against cholangiocellular cancers.

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mutations of the solute carrier family 4 member 1 ( SLC4A1 ) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that afflicts 1 million people annually. This obligate intracellular bacterium boasts one of the largest microbial arsenals of ankyrin repeat-containing protein (Ank) effectors, most of which target the endoplasmic reticulum (ER) by undefined mechanisms. Ank9 is the only one proven to function during infection. Here, we demonstrate that Ank9 bears a motif that mimics the GRIP domain of eukaryotic golgins and is necessary and sufficient for its Golgi localization. Ank9 reaches the ER exclusively by retrograde trafficking from the Golgi. Consistent with this observation, it binds COPB2, a host protein that mediates Golgi-to-ER transport. Ank9 destabilizes the Golgi and ER in a Golgi localization domain-dependent manner and induces the activating transcription factor 4-dependent unfolded protein response. The Golgi is also destabilized in cells infected with O. tsutsugamushi or treated with COPB2 small interfering RNA. COPB2 reduction and/or the cellular events that it invokes, such as Golgi destabilization, benefit Orientia replication. Thus, Ank9 or bacterial negative modulation of COPB2 might contribute to the bacterium's intracellular replication. This report identifies a novel microbial Golgi localization domain, links Ank9 to the ability of O. tsutsugamushi to perturb Golgi structure, and describes the first mechanism by which any Orientia effector targets the secretory pathway., (© 2017 John Wiley & Sons Ltd.)

Mammalian endoplasmic reticulum (ER) exit sites export a variety of cargo molecules including oversized cargoes such as collagens. However, the mechanisms of their assembly and organization are not fully understood. TANGO1L is characterized as a collagen receptor, but the function of TANGO1S remains to be investigated. Here, we show that direct interaction between both isoforms of TANGO1 and Sec16 is not only important for their correct localization but also critical for the organization of ER exit sites. The depletion of TANGO1 disassembles COPII components as well as membrane-bound ER-resident complexes, resulting in fewer functional ER exit sites and delayed secretion. The ectopically expressed TANGO1 C-terminal domain responsible for Sec16 binding in mitochondria is capable of recruiting Sec16 and other COPII components. Moreover, TANGO1 recruits membrane-bound macromolecular complexes consisting of cTAGE5 and Sec12 to the ER exit sites. These data suggest that mammalian ER exit sites are organized by TANGO1 acting as a scaffold, in cooperation with Sec16 for efficient secretion., (© 2017 Maeda et al.)

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1., (© 2017 Gorur et al.)

Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities specifically affecting neoplastic cells without similarly affecting normal cells. Here we performed sequencing-based screening of an shRNA library on a panel of cancer cells of different origins as well as normal cells. The shRNA library was designed to target a subset of genes previously identified using a whole genome screening approach. This focused shRNA library was infected into cells followed by analysis of enrichment and depletion of the shRNAs over the course of cell proliferation. We developed a bootstrap likelihood ratio test for the interpretation of the effects of multiple shRNAs over multiple cell line passages. Our analysis identified 44 genes whose depletion preferentially inhibited the growth of cancer cells. Among these genes ribosomal protein RPL35A, putative RNA helicase DDX24, and coatomer complex I (COPI) subunit ARCN1 most significantly inhibited growth of multiple cancer cell lines without affecting normal cell growth and survival. Further investigation revealed that the growth inhibition caused by DDX24 depletion is independent of p53 status underlining its value as a drug target. Overall, our study establishes a new approach for the analysis of proliferation-based shRNA selection strategies and identifies new targets for the development of cancer therapeutics.

COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β'-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Breed-specific differences in fat tissue accumulation in the pig provide an opportunity to study the genetic background of this process. In the present study three pig breeds, differing in fatness, were analyzed in terms of the size of adipocytes derived from three tissues (subcutaneous, visceral and longissimus dorsi muscle) in relation to transcript levels of genes (SNAP23, BSCL2 and COPA), which encode proteins involved in lipid droplet formation. The analysis of adipocyte size revealed significant effects of breed and tissue and confirmed earlier reports that an elevated backfat thickness in some pig breeds is correlated with a larger adipocyte size. Variability in the transcript abundance of the studied genes among breeds and tissues was observed. We found a positive correlation between the abundance of the SNAP23 transcript and adipocyte diameter. The obtained results indicate that SNAP23 may be considered as an interesting candidate gene involved in adipose tissue growth in the pig., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

Background and Aims: Transport of membranes and proteins in eukaryotic cells is mediated by vesicular carriers. Coatomer complex I (COPI)-coated vesicles are involved in the transport between endoplasmic reticulum (ER) and Golgi complex. Several studies indicated that some subunits of COPI were correlated with the cell proliferation of malignant tumors. The present study focused on the function of coatomer protein complex subunit β 2 (COPB2), one of seven proteins in COPI, in prostate cancer (PCa)., Methods: COPB2 gene expression was first analyzed by immunohistochemistry (IHC) in 15 paired PCa and carcinoma adjacent normal tissue from patients. To investigate the role of COPB2 in PCa, we used lentivirus-mediated small interfering RNA (siRNA) to knockdown COPB2 expression in human PCa cell line PC-3 and assessed it by RT-qPCR. Cellomics ArrayScan VTI imaging and colony formation were conducted to evaluate cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry., Results: COPB2 gene was upregulated in the PCa tissue. Cell proliferation was significantly inhibited in COPB2-silenced PC-3 cells using both Cellomics ArrayScan VTI imaging and colony formation assays. S-phase cell counts were significantly decreased; G1- and G2-phase cell counts were significantly increased in COPB2-siRNA group than the control group. Apoptosis was significantly increased in COPB2-siRNA cells., Conclusions: COPB2 significantly promoted PC-3 cell proliferation and colony formation through the cell cycle and apoptosis pathway. Moreover, COPB2 showed a clinical correlation and may serve as a biomarker for the detection for PCa., (Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.)

Anoctamin-1 (ANO1) is a Ca(2+)-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with β-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of β-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with β-COP in U251 glioblastoma cells, and silencing of β-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that β-COP negatively regulates ANO1 surface expression., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The components involved in cellular trafficking and protein recycling machinery that have been associated with increased Alzheimer's disease (AD) risk belong to the late secretory compartments for the most part. Here, we hypothesize that these late unavoidable events might be the consequence of earlier complications occurring while amyloid precursor protein (APP) is trafficking through the early secretory pathway. We investigated the relevance to AD of coat protein complex I (COPI)-dependent trafficking, an early step in Golgi-to-endoplasmic reticulum (ER) retrograde transport and one of the very first trafficking steps. Using a complex set of imaging technologies, including inverse fluorescence recovery after photobleaching (iFRAP) and photoactivatable probes, coupled to biochemical experiments, we show that COPI subunit δ (δ-COP) affects the biology of APP, including its subcellular localization and cell surface expression, its trafficking, and its metabolism. These findings demonstrate the crucial role of δ-COP in APP metabolism and, consequently, the generation of amyloid-β (Aβ) peptide, providing previously nondescribed mechanistic explanations of the underlying events.

Objectives: Choroid plexus (CP) epithelial cells have multiple functions in the cerebral ventricles, including cerebrospinal fluid (CSF) production and forming part of the blood-CSF barrier. They are also responsible for producing inflammatory mediators involved in meningitis. The present study aimed to elucidate the functions of the CP epithelial cells during CNS inflammation., Materials and Methods: We analyzed the proteome and phosphoproteome in lipid A-treated ECPC-4 mouse CP cells by gel electrophoresis and mass spectrometry., Results: Levels of 10 proteins and seven phosphoproteins were significantly altered by lipid A in time-dependent manners, including V-type proton ATPase subunit B (ATP6V), protein 40 kD, elongation factor-1δ, coatomer subunit ε (COPE), vimentin (isoform CRA a), purine nucleoside phosphorylase, eukaryotic initiation factor-4F splicing variant, put. β-actin, peroxiredoxin-6 isoform 1, and immunoglobulin heavy chain variable region. These proteins could be classified as having cytoskeleton/intermediate filament, protein-folding, signal-transduction, cell-growth, metabolism, and redox-regulation functions. The identified phosphoproteins were HSP 84, γ-actin, HSP 70 cognate, vimentin, tubulin β-4B chain, protein disulfide-isomerase A6 precursor, and heterogenous nuclear ribonucleoprotein, which could be classified as having cytoskeleton/intermediate filament, protein-folding, and metabolism functions., Conclusions: These results indicate that lipid A can change the levels of proteins and phosphoproteins in ECPC-4 cells, suggesting that the identified proteins and phosphoproteins may play important roles in inflammation of the CP.