Proteins In Golgi Apparatus Research Articles

Taurine promotes estrogen synthesis by regulating microRNA-7a2 in mice ovarian granulosa cells

Taurine, acting as a free amino acid, is widely distributed and plays multiple functions, including its regulating effect on estrogen synthesis in ovary. However, the mechanisms of taurine regulating estrogen synthesis in granulosa cells are not well understood. In this study, we identify whether microRNA-7a2 (miR-7a2) is involved in the signaling of taurine regulating estrogen synthesis in mouse granulosa cells for the first time. The results demonstrated that taurine transporter (TauT) co-localized with miR-7a in mouse ovarian granulose cells. Further, taurine treatment markedly enhanced the expression of miR-7a and Cyp19a1 in mouse ovaries and increased serum 17β-estradiol (E2) concentration. Meanwhile, miR-7a2 knockout reversed the effect of taurine on E2. In addition, Golgi apparatus protein 1 (Glg1), a downstream target gene of miR-7a2, was significantly down-regulated by taurine, while Glg1 knockdown markedly increased the Cyp19a1 expression and E2 synthesis. Moreover, taurine affected miR-7a expression via activating p38 signaling. These results suggest that taurine promotes E2 synthesis through p38/miR-7a/Glg1/Cyp19a1 signaling pathway, which is crucial to understand the function and mechanism of taurine on estrogen synthesis.

MicroRNA-7a2 Contributes to Estrogen Synthesis and Is Modulated by FSH via the JNK Signaling Pathway in Ovarian Granulosa Cells.

MicroRNA-7a2 (miR-7a2) plays fundamental roles in the female reproductive axis, and estrogen is indispensable for maintaining ovary function. However, the interaction between miR-7a2 and ovarian function is unclear. The present study aimed to determine whether and how miR-7a2 functions in estrogen synthesis. Firstly, the results verified that miR-7a was highly expressed in ovarian granulosa cells. The knockout (KO) of miR-7a2 caused infertility and abnormal ovarian function in mice. Concomitantly, the Cyp19a1 expression and estrogen synthesis were significantly inhibited, which was validated in primary granulosa cells. The mice transplanted with miR-7a2 KO ovaries showed similar results; however, estrogen supplementation reversed infertility. In the in vitro experiment, follicle-stimulating hormone (FSH) significantly improved the expression of miR-7a and Cyp19a1 and the synthesis of estrogen. However, the miR-7a2 KO markedly reversed the function of FSH. Also, FSH upregulated miR-7a by activating the (c-Jun N-terminal kinase) JNK signaling pathway. In addition, Golgi apparatus protein 1 (Glg1) was shown to be the target gene of miR-7a2. These findings indicated that miR-7a2 is essential for ovarian functions with respect to estrogen synthesis through the targeted inhibition of the expression of Glg1 and then promoting Cyp19a1 expression; the physiological process was positively regulated by FSH via the JNK signaling pathway in granulosa cells.

Deciphering cardiac O-GlcNAcylation: A new perspective of cardiac mitochondrial regulation

Mitochondria is a key cellular component. During pathophysiological stress mitochondrial function is regulated dynamically to provide cell with sufficient energy. O-GlcNAcylation, a post-translational modification associated with stress response, has been linked to beneficial effect when stimulated in shock situation, yet the link between O-GlcNAc and mitochondrial function remains poorly understood. To evaluate mitochondrial impact of O-GlcNAc modulation. Hearts were harvested from Wistar rat from Ctrl, shocked (LPS) and treated by NButGT, a pharmacological O-GlcNAcylation inducer. Respiration rate was assessed polarographically in isolated mitochondria. O-GlcNAcylomic was performed by mass spectrometry on cardiac tissue from rats at D0 (after birth) and D28 (suckling weaning) as well as from LPS and NButGT treated animals. Gene Ontology (GO) for cellular components (CC) of identified cardiac O-GlcNAcylated proteins from both groups were obtained by running the clusterProfileR package. Within D0/28 groups, 647 O-GlcNAcylated proteins were found and 1012 in Ctrl/LPS/NButGT groups. Protein for subsequent analysis were selected by the log(FoldChange) > 2 or < − 2 with the following ratio: D28/D0 or LPS/Ctrl and LPS/NButGT. GO enrichment analysis revealed that O-glacNacyltion of, endoplasmic reticulum, mitochondria, and Golgi apparatus proteins was greater at D28. In the LPS model mitochondria and endoplasmes reticulum ranked as the top two organelles affected by NBuGT Moreover, induction of O-GlcNAcylation in isolated mitochondria rapidly stimulateed maximal mitochondrial respiration specifically in presence of Complex I substrates suggesting that O-GlcNAcylation dynamically regulate OXPHOS. Mitochondrial O-GlcNAcylation appears to play a major role in pathophysiology. Targeting mitochondrial O-GlcNAcylation could represent a new therapeutic challenge and should be further explored.

MALDI-TOF mass spectrometric protein profiling of microvesicles produced by the NK-92 natural killer cell line

Extracellular vesicles that are shed from the plasma membrane contain a wide range of molecules, among which are proteins, lipids, nucleic acids, and sugars. The cytotoxic proteins of natural killer cells play a key role in the implementation of their cytolytic functions. One of the important steps in understanding the distant communication of cells is the determination of the proteome of microvesicles. This study was aimed at the protein profiling of the microvesicles produced by the NK-92 natural killer cell line. 986 proteins with a variety of functions were identified in the lysate of microvesicles using the MALDI-TOF mass spectrometric analysis. With automated methods of functional analysis applied, it has been shown that the largest protein groups are hypothetical proteins, proteins with unknown functions, and domains. The most representative groups are also comprised by transcription regulators; intracellular signaling proteins; RNA translation, transcription, processing, and utilization regulators; receptors; protein processing and proteolysis regulators; amino acid metabolism enzymes, as well as transport proteins and transport regulators. Minor functional groups are represented by vitamins and mineral metabolism enzymes, membrane and microdomain-forming proteins, hormones, hemostatic regulators, regulators of sensory systems, specific mitochondrial and Golgi apparatus proteins, and extracellular signaling proteins. An intermediate position is occupied by various functional groups, including cytoskeleton and motor proteins; proteins of centrioles; ion channels and their regulators; proteins of the ubiquitin-proteasome pathway of protein degradation; lipid, steroid, and fatty acid metabolism enzymes; nucleic acid base and carbohydrate metabolism enzymes, as well as energy metabolism enzymes and other proteins involved in intermediate metabolism; proteins of the immune response and inflammation; antigens and histocompatibility proteins; cytokines and growth factors; regulators of apoptosis, autophagy, endocytosis, and exocytosis; regulators of the cell cycle and division; regulators of proliferation, cell differentiation, and morphogenesis; regulators of cell adhesion and matrix metabolism; nuclear transport proteins; transposition proteins; DNA replication and repair proteins, as well as inactive proteins. The data obtained expand the existing knowledge of the distant communication of cells and indicate new mechanisms of interaction between natural killer and target cells.

Computational Proteome-Wide Study for the Prediction of Escherichia coli Protein Targeting in Host Cell Organelles and Their Implication in Development of Colon Cancer.

Enterohemorrhagic Escherichia coli infection is associated with gastrointestinal disorders, including diarrhea and colorectal cancer. Although evidences have established the involvement of E. coli in the growth of colon cancer, the molecular mechanisms of carcinogenesis of cancer growth and development are not well understood. We analyzed E. coli protein targeting in host cell organelles and the implication in colon cancer using in silico approaches. Our results indicated that many E. coli proteins targeted the endoplasmic reticulum (ER), ER membranes, Golgi apparatus, Golgi apparatus membranes, peroxisomes, nucleus, nuclear membrane, mitochondria, and mitochondrial membrane of host cells. These targeted proteins in ER, Golgi apparatus, peroxisomes, nucleus, and mitochondria may alter the normal functioning of various pathways including DNA repair, apoptosis, replication, transcription, and protein folding in E. coli-infected host cells. The results of the current in silico study provide insights into E. coli pathogenesis and may aid in designing new preventive and therapeutic strategies.

Activity of the SPCA1 Calcium Pump Couples Sphingomyelin Synthesis to Sorting of Secretory Proteins in the Trans-Golgi Network

How the principal functions of the Golgi apparatus-protein processing, lipid synthesis, and sorting ofmacromolecules-are integrated to constitute cargo-specific trafficking pathways originating from the trans-Golgi network (TGN) is unknown. Here, we show that the activity of the Golgi localized SPCA1 calcium pump couples sorting and export of secreted proteins to synthesis of new lipid in the TGN membrane. A secreted Ca2+-binding protein, Cab45, constitutes the core component of a Ca2+-dependent, oligomerization-driven sorting mechanism whereby secreted proteins bound to Cab45 are packaged into a TGN-derived vesicular carrier whose membrane is enriched in sphingomyelin, a lipid implicated in TGN-to-cell surface transport. SPCA1 activity is controlled by the sphingomyelin content of the TGN membrane, such that local sphingomyelin synthesis promotes Ca2+ flux into the lumen of the TGN, which drives secretory protein sorting and export, thereby establishing a protein- and lipid-specific secretion pathway.

Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling.

Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

Abstract P4-09-23: Gene expression signatures of microcalcifications among Taiwanese breast cancers

Abstract Introduction: Microcalcification is one of the most common radiological and pathological features of breast ductal carcinoma in situ (DCIS), and to a lesser extent, invasive breast cancer. In current study, we evaluated the transcriptional profiles associated with the phenomenon of ectopic mammary mineralization and a gene expression signature is derived. Materials and methods: a total of 109 consecutive breast invasive cancers were prospectively collected and assayed with Affymetrix Human Genome U133 Plus 2.0 microarrays. The presence of microcalcification was confirmed by histopathological examinations as well as reviews of pre-operative mammography. The associations of gene expression profiles with microcalcifications and relevant clinical features such as DCIS including both comedo/high-grade and non-comedo subtypes were tested. Results: Microcalcifications were presented in 84 (80%) of the study population as confirmed by pathological examination. Of these 84 patients, 81 (96%) were grown with coexistent DCIS microscopically, while only 8 (38%) of the 21 patients without concurrent microcalcifications, the invasive tumors were accompanied with DCIS (Chi-square test, P&amp;lt;0.001). In addition, high-grade (comedo) type DCIS were presented in 44 (54%) of the 81 cancers with microcalcifications whereas only 15% (n=2) of tumors with DCIS but without microcalcificaitons were of high-grade (comedo) type. There were 69 genes differentially expressed between breast cancers with and without microcalcifications (nominal P&amp;lt;0.001 with 10,000 random permutations), and 11 were associated with high-grade (comedo) type DCIS including APOD, CCDC183, SLMO1, SLC6A5, FMO1, QPRT and CES4A. The enriched Gene Ontology categories encompasses glycosaminoglycan, aminoglycan metabolic processes, Golgi apparatus cellular component and protein ubiquitination, indicating an active secretory process. The intersect (18 probesets) of microcalcificaion and DCIS-associated genes provided the best predictive accuracy of 82% with Bayesian compound covariate predictor. Performance of compound covariate predictor classifier:ClassSensitivitySpecificityPPVNPVWith microcalcification0.8260.2170.7980.25Without microcalcification0.2170.8260.250.798 Compared with mammography alone, the diagnostic accuracy of gene expression-based signature is much improved (cross-validated ROC AUC: 0.738). Discussion: Our study suggested that mammary microcalcification is not only the earlier detectable radiological finding for disease screening but the phenomenon itself resulted from distinct biological processes that constituted the molecular heterogeneity of human breast cancers. Further studies to evaluate the prognostic significance of microcalcifications are warranted. Citation Format: Huang C-C, Huang C-S, Tu S-H, Tsai M-L. Gene expression signatures of microcalcifications among Taiwanese breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-23.

Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer.

Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers.

UNC50 prompts G1/S transition and proliferation in HCC by regulation of epidermal growth factor receptor trafficking.

BackgroundUNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans, but little is known about UNC50 gene function in human biology despite it being conserved from yeast to high eukaryotes.ObjectivesWe investigated the relation between UNC50 and human hepatocellular carcinoma (HCC) and the potential mechanisms underlying HCC development.Methods UNC50 mRNA expression patterns in 12 HCC and adjacent non-cancerous tissues determined using northern blotting were confirmed by real-time PCR in another 44 paired tissues. Microarray experiments were used to screen for global effects of UNC50 knockdown in the Hep3B cell line, and were confirmed by real-time PCR, western blotting, flow cytometry, and tetrazolium assay in both UNC50 overexpression and knockdown Hep3B cells.ResultsUNC50 expression levels were upregulated in HCC tissues in comparison with the adjacent non-cancerous tissues. UNC50 knockdown reduced mRNA levels of the downstream targets of the epidermal growth factor receptor (EGFR) pathway: cyclin D1 (CCND1), EGF, matrix metalloproteinase-7 (MMP7), aldose reductase-like 1 (AKR1B10), cell surface–associated mucin 1 (MUC1), and gastrin (GAST). Moreover, UNC50 influenced EGF, inducing cell cycle entry by affecting cell surface EGFR amounts.Conclusions UNC50 may plays some roles in HCC progression by affecting the EGFR pathway.

Coat protein I depletion-associated Golgi fragmentation in an Alzheimer's disease model

The onset and progression of Alzheimer's disease (AD) is closely associated with amyloid β (Aβ) peptide-induced cytotoxicity and abnormal protein transportation caused by breakdown of endoplasmic reticulum (ER)–Golgi apparatus trafficking network. Although the fragmentation of Golgi apparatus has been reported in AD human patients and various AD model animals, the molecular mechanisms causing the morpho-functional impairments of Golgi apparatus during AD progression remain poorly understood. Thus, we investigated the ultrastructure of Golgi apparatus and coat protein I (COPI) expression in β-amyloid precursor protein/presenilin-1 double transgenic mouse and Aβ-stimulated BV-2 cell as an AD model using cryo-immunogold electron microscopy. In the neurons of the hippocampus of transgenic mouse and BV-2 cell, the cisternae of Golgi stacks were fragmented in difference with that of wild type mouse and cells. In addition, we further showed the COPI depletion in Golgi apparatus, which demonstrated the impairment of molecular integrities of Golgi apparatus. Taken together, our results provide insights into the Golgi apparatus-involved morphopathology of AD and we suggest that the Golgi fragmentation is caused by the depletion of COPI affecting the intra-Golgi transport through stimulation and accumulation of Aβ during AD development.

Regulator of calcineurin 1-1L protects cardiomyocytes against hypoxia-induced apoptosis via mitophagy.

Mitochondrial dysfunction induced by myocardial ischemia is the primary cause of cardiac cell death. Specific removal of damaged mitochondria through mitophagy may be beneficial for cardiomyocyte protection against ischemia. Regulator of calcineurin 1-1L (Rcan1-1L) has been implicated in mitophagy induction in neurons. However, whether or not Rcan1-1L can evoke mitophagy in cardiomyocytes during hypoxia remains unknown. This study aims to investigate the effect of Rcan1-1L overexpression on cardiomyocytes during hypoxia and the possible underlying mechanism. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Rcan1-1L overexpression inhibited cell growth under normoxic conditions, whereas Rcan1-1L overexpression significantly reversed the growth inhibition induced by hypoxia. The results of terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling assay showed that cell apoptosis induced by hypoxia was markedly reduced by Rcan1-1L overexpression. In addition, Rcan1-1L overexpression inhibited the expression of the proapoptotic protein Bcl-2-associated death promoter and increased that of the antiapoptotic protein Bcl-2. Rcan1-1L overexpression opened the mitochondrial permeability transition pore and decreased mitochondrial mass. Meanwhile, the release of reactive oxygen species from mitochondria was suppressed by Rcan1-1L. Autophagy flow activation represented by mammalian target of rapamycin inhibition and microtubule-associated protein light chain 3 (LC3) upregulation was also demonstrated. Compared with endoplasmic reticulum and Golgi apparatus protein markers, the mitochondrial protein marker translocase of outer mitochondrial membranes 20 (TOM20) was downregulated by Rcan1-1L overexpression. Moreover, Rcan1-1L increased mitophagy receptor Parkin translocation into mitochondria from cytosol. Additionally, the effect of Rcan1-1L on cell growth, cell apoptosis and mitochondria mass was blocked by Parkin expression silencing. Overall, these data suggest that Rcan1-1L protects cardiomyocytes from hypoxia-induced apoptosis by inducing mitophagy partly through Parkin. This study provided novel insights into the prevention and treatment of ischemic heart disease.

Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

ABSTRACTThe replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion.IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.

Topological Regulation of Lipid Balance in Cells

Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient.

Differential expression analysis of Golgi apparatus proteomes in hepatocellular carcinomas and the surrounding liver tissues

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Liver is the largest human digestive gland with abundant Golgi apparatus involved in cell division, migration and apoptosis and others. In the present study, Golgi apparatus of HCC and the surrounding liver tissues were isolated by sucrose density gradient centrifugation and identified by electron microscopy and enzymology methods. Using 2-D gel electrophoresis and mass spectrometry, 17 differentially expressed protein of Golgi apparatus in HCC and the surrounding liver tissue were screened and identified in the Mascot database. Of those differentially expressed proteins, six were upregulated and 11 were downregulated, some of them were related to the biological processes such as protein sorting, glycosylation, cell cycle regulation, transcription regulation and Golgi integrity. One protein, annexin A5, was verified to be upregulated in HCC by western blot. The differentially expressed proteins may provide new insight into HCC biology and potential diagnostic and therapeutic biomarkers.

Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques

Patients with Parkinson's disease show persistent microglial activation in the areas of the brain where the degeneration of dopaminergic neurons takes place. The reason for maintaining this activated state is still unknown, but it is thought that this persistent microglial activation may contribute to the degeneration of dopaminergic neurons. In this study, we report the microanatomical details of microglia and the relationship between microglia and neurons in the substantia nigra pars compacta of Parkinsonian monkeys years after insult with MPTP. We observed that microglial cells appear polarized toward dopaminergic neurons in MPTP-treated macaques compared to untreated animals and present clear phagocytic characteristics, such as engulfing gliaptic contacts, an increase in Golgi apparatus protein machinery and ball-and-chain phagocytic buds. These results demonstrate that activated microglia maintain phagocytic characteristics years after neurotoxin insult, and phagocytosis may be a key contributor to the neurodegenerative process.

Abstract 4377: The Golgi protein GLG1 coordinates ARF3 activation in tumor cells.

Abstract Increasing evidence suggests that endocytosis and membrane trafficking are deregulated in cancer cells. Because of their dependence on selected signaling pathways, tumor cells may need to rely on accelerated receptor recycling and increased secretion of a variety of molecules such as matrix components, adhesion molecules and growth factors. Mass spectrometry analysis of proteins associated with separase, recently shown to be an important regulator of membrane trafficking, identified the Golgi apparatus protein 1 (GLG1). GLG1 is primarily known as an E-selectin ligand that mediates the initial step of tethering and rolling of leukocytes on vascular endothelium. We therefore addressed the possibility that GLG1 could play a similar role in circulating tumor cells, facilitating arrest and extravasation into adjacent tissues. Consistent with this possibility, tail vein injection of stably GLG1-depleted tumor cells into NOD/SCID mice resulted in reduced numbers of metastatic nodules. Moreover, metastatic tumor growth was almost exclusively intravascular, indicating impaired extravasation of tumor cells. Further investigation, however, showed that, in tumor cells, GLG1 is almost exclusively localized to the Golgi apparatus, challenging the notion that it plays a direct role in mediating tumor cell adhesion to endothelium as in leukocytes. To elucidate the molecular mechanisms by which GLG1 could affect tumor cell migration through interactions at the Golgi level, we conducted various experiments using GLG1-depleted human tumor cell lines. Transient silencing of GLG1 by siRNA was observed to modify cell shape (elongated morphology), to reduce migration of a range of tumor cell lines and to markedly alter Golgi architecture. Interestingly, subsequent experiments led to the identification of a new interactor of GLG1, the Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), an ARF-GEF required to initiate membrane vesicle formation at the trans-Golgi compartment by promoting guanine-nucleotide exchange on ARF1 and ARF3. GLG1 was shown to participate in BIG1 and ARF3 recruitment to the Golgi membrane and subsequent ARF3 activation. Surprisingly, the observed morphological disruption of the Golgi apparatus and reduced ARF3 activation did not affect the secretory pathway in general, but rather seemed to impair secretion of selected molecules including MMP-9. Taken together, our observations provide insight into a novel and specific role of GLG1 in tumor progression by affecting, thanks to its privileged position in the secretory pathway, trafficking of key molecules implicated in cell migration. Citation Format: Anne Planche. The Golgi protein GLG1 coordinates ARF3 activation in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4377. doi:10.1158/1538-7445.AM2013-4377

Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.