scholarly journals Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

1994 ◽  
Vol 14 (4) ◽  
pp. 2419-2428 ◽  
Author(s):  
H H van Es ◽  
S Karcz ◽  
F Chu ◽  
A F Cowman ◽  
S Vidal ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Plasmodium Falciparum Malaria ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Malaria Parasites ◽  
Pfmdr1 Gene ◽  
Alpha Galactosidase

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine

1994 ◽  
Vol 14 (4) ◽  
pp. 2419-2428
Author(s):  
H H van Es ◽  
S Karcz ◽  
F Chu ◽  
A F Cowman ◽  
S Vidal ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Plasmodium Falciparum Malaria ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Malaria Parasites ◽  
Pfmdr1 Gene ◽  
Alpha Galactosidase

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 717-717
Author(s):  
Nithya Krishnan ◽  
Jeff R. Bailey ◽  
Victoria Summey-Harner ◽  
Claudio Brunstein ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Philadelphia Chromosome ◽  
Protein Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

scholarly journals A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

1993 ◽  
Vol 123 (3) ◽  
pp. 759-766 ◽  
Author(s):  
D C Love ◽  
J D Esko ◽  
D M Mosser
Keyword(s):  
Heparan Sulfate ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Activity ◽  
Heparan Sulfate Proteoglycans ◽  
Heparin Binding ◽  
Wild Type ◽  
Amastigote Form ◽  
High Affinity ◽  
Leishmania Amastigotes

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

scholarly journals Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 991-999 ◽  
Author(s):  
M Watarai ◽  
S Funato ◽  
C Sasakawa
Keyword(s):  
Epithelial Cells ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Shigella Flexneri ◽  
Chinese Hamster ◽  
Invasive Capacity ◽  
Beta 1 Integrin ◽  
Cellular Components

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

TFPI-Beta Is An Effective Inhibitor of TF-FVIIa Mediated Coagulation and Tumor Cell Invasion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 347-347
Author(s):  
Susan A Maroney ◽  
Josephine P Ferrel ◽  
Alan E. Mast
Keyword(s):  
Cell Surface ◽  
Inhibitory Activity ◽  
Cho Cells ◽  
Potent Inhibitor ◽  
Terminal Region ◽  
Solution Phase ◽  
Effective Inhibitor ◽  
Wild Type

Abstract Abstract 347 Tissue Factor Pathway Inhibitor (TFPI) is the primary physiological inhibitor of TF-fVIIa, the in vivo activator of blood coagulation. TFPI is an alternatively spliced protein with two major isoforms, TFPI-alpha and TFPI-beta. These isoforms differ in their C-terminal domain structure and their mechanism for cell surface attachment with TFPI-alpha indirectly associating with the endothelial surface through binding to a GPI-floated protein while TFPI-beta is directly attached to a GPI-float. In addition, the isoforms are differentially expressed in mouse tissues, with TFPI-alpha present in placenta, embryo and platelets, while TFPI-beta is the predominant isoform in adult vascular beds. However, TFPI-beta has ∼20-fold decreased activity when compared to TFPI-alpha in solution phase plasma clotting assays. We hypothesized that TFPI-beta will have more physiologically relevant activity when associated with a cell surface instead of in solution phase. To test this hypothesis, a CHO cell system that allows measurement of the anti-TF activity of different cell surface forms of TFPI in both in vitro and in vivo assays was developed. CHO cells were stably transfected with TF creating CHO-TF cells. In contrast to wild type CHO cells, CHO-TF cells 1) generate fXa in vitro in the presence of fVIIa, fX and calcium ions, 2) migrate through matrigel in transwell assays, and 3) produce tumors in the lungs of SCID mice following tail vein injection. The ability of solution phase TFPI-alpha and TFPI-beta to inhibit TF-fVIIa mediated fXa generation on the surface of the CHO-TF cells was examined with amidolytic assays. The Ki(final) for TFPI-alpha and TFPI-beta were 5.81 nM and 21.6 nM, respectively, confirming that solution phase TFPI-beta has decreased inhibitory activity. To examine the activity of cell surface associated TFPI-beta, CHO-TF cells were co-transfected with either TFPI-beta or equal amounts of an altered form of TFPI, called K1K2K3-GPI that is similar to TFPI-alpha but lacks its basic C-terminal region. Forms of TFPI containing the basic C-terminal region with a GPI-float could not be studied because they were not expressed by the CHO cells. CHO-TF cells expressing TFPI-beta had equal inhibitory activity to that observed in CHO-TF cells with K1K2K3-GPI suggesting that TFPI-beta is a potent inhibitor of TF activity when associated with the cell surface. The activity of cell surface associated TFPI-beta was further examined in transwell migration assays. The number of cells migrating through matrigel in multiple 20X fields was averaged. In this assay, cell surface associated TFPI-beta was a potent inhibitor of TF activity. The results are as follows: wild type CHO cells—6.6+/−4.1; CHO-TF cells—98.0+/−32.5; CHO-TF cells with TFPI-beta—9.05+/−7.0; CHO-TF cells with K1K2K3-GPI—43.5+/−21.5. Migration of CHO-TF cells was blocked using argatroban, an active site directed inhibitor of thrombin (15.0+/−4.5 CHO-TF cells migrated in the presence of 100 micromolar argatroban), demonstrating that the likely mechanism for the TF-mediated cell migration is generation of thrombin with cellular activation through cleavage of protease activated receptors. In the SCID tumor model, the severity of lung tumor burden was graded as 1–4 by a pathologist blinded to the cell type injected, n=5-8 per group. Average scores were: wild type CHO cells—1.17; CHO-TF cells 2.80; CHO-TF cells with TFPI-beta—2.14; demonstrating that cell surface associated TFPI-beta is an effective inhibitor of TF activity in vivo. Thus, while TFPI-beta has limited anticoagulant activity when examined in solution phase assays in vitro, evaluation of cell surface associated TFPI-beta reveals that it is a highly effective inhibitor of TF-fVIIa activity both in vitro and in vivo. Disclosures: Mast: Novo Nordisk: Research Funding; Siemens: Speakers Bureau.

scholarly journals Farnesyl Diphosphate Synthase Is a Cytosolic Enzyme in Leishmania major Promastigotes and Its Overexpression Confers Resistance to Risedronate

2006 ◽  
Vol 5 (7) ◽  
pp. 1057-1064 ◽  
Author(s):  
Aurora Ortiz-Gómez ◽  
Carmen Jiménez ◽  
Antonio M. Estévez ◽  
Juana Carrero-Lérida ◽  
Luis M. Ruiz-Pérez ◽  
...  
Keyword(s):  
Drug Target ◽  
Leishmania Major ◽  
Intracellular Localization ◽  
Initial Step ◽  
Isoprenoid Biosynthesis ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type

ABSTRACT Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.

scholarly journals TFIIA Plays a Role in the Response to Oxidative Stress

2006 ◽  
Vol 5 (7) ◽  
pp. 1081-1090 ◽  
Author(s):  
Susan M. Kraemer ◽  
David A. Goldstrohm ◽  
Ann Berger ◽  
Susan Hankey ◽  
Sherry A. Rovinsky ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Expression Profiles ◽  
Intracellular Localization ◽  
Regulation Of Gene Expression ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.

Sign in / Sign up

Export Citation Format

Share Document