The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1975-1989 ◽  
Author(s):  
T. Nilsson ◽  
C. Rabouille ◽  
N. Hui ◽  
R. Watson ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Cell Lines ◽  
Hela Cells ◽  
Kin Recognition ◽  
Dramatic Effect ◽  
Golgi Stack ◽  
Stable Cell Lines ◽  
Membrane Spanning ◽  
Necessary And Sufficient

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 989-989 ◽  
Author(s):  
Abdullah M. Ali ◽  
Thiyam R. Singh ◽  
Ruhikanta A. Meetei
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Affinity Purification ◽  
Gene Products ◽  
Core Complex ◽  
Purification Method

Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.

Mutations Identified in Thrombocytopenia THC2 Are Likely to Dysregulate ANKRD26 Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 708-708
Author(s):  
Chiara Gnan ◽  
Patrizia Noris ◽  
Felisa C. Molinas ◽  
Shinji Kunishima ◽  
Paula Graciela Heller ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hela Cells ◽  
Luciferase Activity ◽  
Luciferase Assay ◽  
Published Data ◽  
Bleeding Tendency ◽  
Wild Type ◽  
Significant Difference ◽  
Gene Assay

Abstract Abstract 708 Thrombocytopenia 2 (THC2, MIM 188000) is an autosomal dominant form of thrombocytopenia described for the first time in a large Italian family, which allowed us to map a locus on 10p11.1-p12. Since the recent identification of ANKRD26 (Pippucci et al, Am J Hum Genet 88:115, 2011), as the gene responsible for the disease, the screening led to recognition of 25 families (91 patients). Although most families were of Italian origin, patients were also from North America, Argentina, Senegal, Japan and Spain, indicating that the disease is distributed worldwide. Confirming recently published data (Noris et al, Blood 117:6673, 2011), this cohort showed that thrombocytopenia was moderate and bleeding tendency was usually mild. Most patients were characterized by deficiency of both glycoprotein Ia and a-granules but normal platelet aggregation in vitro. Bone marrow examination and serum thrombopoietin levels were indicative of dysmegakaryopoiesis. Moreover, there was evidence of leukemia risk in patients with ANKRD26 mutations. The 12 mutations identified so far are all localized in a short stretch of 22 nucleotides of the 5' untranslated region (5'UTR). The effect of three mutations was evaluated in a reporter gene assay with the luciferase gene under the control of the wild type and mutated 5'UTR. When constructs were transfected in K562 and undifferentiated DAMI cell lines, no significant difference in luciferase activity was observed. However, when the constructs were transfected in megakaryocytes obtained from differentiation of the DAMI cells, a significant increase in activity was reported, suggesting that the 5'UTR plays a regulatory role of the ANKRD26 expression during megakaryopoiesis (Pippucci et al, Am J Hum Genet 88:115, 2011). To further investigate the mechanisms responsible for the ANKRD26 expression, we tested the activity of a putative regulatory region (730 bp), containing 574 bp upstream of the transcription initiation site and the 5'UTR. The effect of two mutations (c.-128G>A and c.-116C>T) was evaluated in both orientations using a luciferase assay in HeLa cells. There was no significant difference between wild type and mutated inserts cloned in either sense or antisense orientation, though there was a slight transcriptional increase of the sense mutated constructs and a reduction of the antisense mutated sequences in comparison with the wild type inserts. In order to test the promoter region in a more suitable model, the same constructs were transfected in DAMI differentiated megakaryocytes. Whereas the antisense wild type and mutated constructs did not show any transcriptional activity, the sense mutated constructs generated a statistically significant increase of activity, suggesting that the mechanisms controlling the expression of ANKRD26 are different in the two cell lines. The role of 5'UTR was further investigated by testing a fragment (574 bp) in which 5'UTR was deleted. In megakaryocytes, this sequence generated a statistically significant increase of the luciferase activity compared to the 730 bp insert. On the contrary, the same constructs transfected in Hela cells resulted in a reduction of the luciferase activity. From these preliminary data we hypothesize that the 5'UTR region plays an important role inhibiting the ANKRD26 expression during megakaryopoiesis through the binding of factors, whose recognition sites are destroyed by mutations identified in patients. Consistent with this hypothesis, ANKRD26 is expressed in human CD34+ and BFU-E but hardly detectable in CD41+ cells. Disclosures: No relevant conflicts of interest to declare.

Determination of the cytotoxic activity of Campylobacter strains isolated from bovine and swine carcasses in north-eastern Poland

2015 ◽  
Vol 18 (3) ◽  
pp. 579-586 ◽  
Author(s):  
B. Wysok ◽  
J. Uradziński ◽  
J. Wojtacka
Keyword(s):  
Cytotoxic Activity ◽  
Cell Lines ◽  
Hela Cells ◽  
Pcr Analysis ◽  
High Prevalence ◽  
North Eastern ◽  
One Year ◽  
Human Infections

Abstract The study was carried out to determine the cytotoxin production by Campylobacter spp. isolated from slaughtered cattle and swine in north-eastern Poland. In total three commercial slaughterhouses were sampled during one year. Carcass swabs were taken to detect the level of Campylobacter spp. contamination. Campylobacter spp. was found in 50 (34%) out of 147 swine carcasses examined. PCR analysis revealed 4 (8%) isolates to be C. jejuni, and 46 (92%) to be C. coli. From a total of 373 bovine carcasses, Campylobacter spp. were isolated from 49 (13.1%) samples. The results regarding the occurrence of cdt genes associated with cytotoxicity indicated that 100% of C. jejuni and 67.4% C. coli obtained from pigs had all three cdtA, cdtB and cdtC genes. In case of C. jejuni strains isolated from cattle all cdt genes were confirmed in 93.9% isolates. The isolates possessesing all cdt genes had higher cytotoxic activity against cell lines used. The isolates both from cattle and swine were characterized by the highest cytotoxicity against HeLa cells. The values obtained reached 80.8% for C. jejuni isolates from cattle and 76.2% for C. jejuni and 69.0% for C. coli isolates from swine. High prevalence of cytotoxicity in Campylobacter spp. indicates a significant epidemiological role of this pathogen in human infections.

scholarly journals The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner

2002 ◽  
Vol 76 (1) ◽  
pp. 105-117 ◽  
Author(s):  
Dimiter G. Demirov ◽  
Jan M. Orenstein ◽  
Eric O. Freed
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Virus Replication ◽  
Hela Cells ◽  
Virus Release ◽  
Cell Type ◽  
Particle Release ◽  
Immunodeficiency Virus ◽  
T Cell Lines

ABSTRACT The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.

scholarly journals Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Sujit Pujhari ◽  
Tayyba T. Baig ◽  
Alexander N. Zakhartchouk
Keyword(s):  
Cell Lines ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Intermediate Step ◽  
Structural Protein ◽  
Pork Industry ◽  
Stable Cell Lines ◽  
Apoptosis Inhibition ◽  
Induced Apoptosis

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells. In addition, we found significantly higher activities of transcriptional factors (NF-κB and AP-1) in both cell lines as compared to MARC-145 (parent cells). Overall, our data suggest that, although both stable cell lines activate NF-κB and AP-1, GP2 triggers the antiapoptotic process through an intermediate step that needs to be further investigated.

Validation of the Function of 14-3-3 ζ in Multiple Myeloma (MM)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1369-1369
Author(s):  
Yanyan Gu ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Stable Cell Lines ◽  
Induced Apoptosis ◽  
The Impact

Abstract Abstract 1369 The 14-3-3 protein family includes seven members, β, γ, ε, η, σ, τ and ζ. With over 200 binding partners, 14-3-3 proteins act as integrators of diverse cell signaling pathways and participate in metabolism, cell cycle regulation, survival and apoptosis. 14-3-3ζ has been implicated in many cancers such as hepatocellular carcinoma, gastric cancer, breast cancer, lung carcinoma and lymphoma. However, the role of 14-3-3ζ in MM has not been extensively explored. Preliminary data from an affymatrix GEP profile of normal plasma cells (NPC), MGUS, Smoldering myeloma (SM) or multiple myeloma (MM) demonstrates statistically increased expression of 14-3-3 ζ in the transition between MGUS and SM. Among patients with newly diagnosed symptomatic MM, 14-3-3 ζ expression appears to be higher in the higher risk genetic subsets. These data suggest 14-3-3ζ plays a prominent role in the biology of MM especially among high risk myeloma patients. In order to identify the impact of 14-3-3 ζ signaling on MM proliferation and survival, we developed 14-3-3ζ silenced and over expressing stable cell lines to interrogate the biological role of 14-3-3ζ in MM. Using a library of human MM cell lines, we found that 14-3-3ζ is universally expressed in all MM cell lines examined. Knockdown of 14-3-3ζ significantly inhibits cell growth and proliferation in LP1 and U266 cells, which is partly related to G1 cell cycle arrest. Relevant signaling proteins such as Mcl-1, Bcl2, phospho-Akt and CDK6 decrease after silencing 14-3-3ζ. Furthermore, we performed gene expression profiling of LP1 scrambled and knockdown stable cell lines in order to identify key changes in gene regulation that may be mediated via 14-3-3ζ. The GEP data suggests that 14-3-3ζ is responsible for but not limited to several important signaling pathways, such as glycolysis/gluconeogenesis, p53 Signaling, NRF2-mediated oxidative stress response and death receptor signaling. In addition, we evaluated the effect of 14-3-3ζ expression on the drug sensitivity to commonly used chemotherapeutic compounds in MM treatment, such as bortezomib, etoposide, dexamethasone, melphalan, lenalidomide, doxorubicin and romidepsin. Knockdown 14-3-3ζ sensitizes cells to romidepsin- induced apoptosis, as demonstrated by Annexin V staining and western blot assay for caspase cleavage. However, bortezomib- induced apoptosis is significantly inhibited when 14-3-3ζ is silenced. Bortezomib (5nM)-induced apoptosis decreased from 37% in LP1 cells expressing shRNA with scrambled sequence to 14% in LP1 cells where 14-3-3 ζ is silenced. Moreover, 14-3-3ζ knockdown effectively inhibits bortezomib induced NOXA upregulation, suggesting a possible new molecular mechanism for the effects of 14-3-3ζ in bortezomib mediated apoptosis. Taken together, our work reveals the important biological function of 14-3-3ζ in MM growth, survival and proliferation; the data also provides valuable information for the development of new therapeutic strategies facilitating drug sensitivity and overcoming drug resistance. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Merck: Consultancy.

Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

1998 ◽  
Vol 111 (1) ◽  
pp. 45-60 ◽  
Author(s):  
S. Rottger ◽  
J. White ◽  
H.H. Wandall ◽  
J.C. Olivo ◽  
A. Stark ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Immunoelectron Microscopy ◽  
Hela Cells ◽  
Subcellular Fractionation ◽  
Transferase Activity ◽  
Medial Part ◽  
Membrane Structures ◽  
Golgi Stack ◽  
Vesicular Membrane

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.

Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides

1995 ◽  
Vol 108 (4) ◽  
pp. 1617-1627 ◽  
Author(s):  
C. Rabouille ◽  
N. Hui ◽  
F. Hunte ◽  
R. Kieckbusch ◽  
E.G. Berger ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Hela Cells ◽  
Stable Expression ◽  
Hela Cell Line ◽  
Specific Antibodies ◽  
Hela Cell Lines ◽  
Golgi Network

The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.

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