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 In Vitro Antileishmanial Activity of Nicotinamide

2005 ◽  
Vol 49 (2) ◽  
pp. 808-812 ◽  
Author(s):  
D. Sereno ◽  
A. Monte Alegre ◽  
R. Silvestre ◽  
B. Vergnes ◽  
A. Ouaissi
Keyword(s):  
Leishmania Major ◽  
Growth Arrest ◽  
Antileishmanial Activity ◽  
Vitamin B ◽  
Wild Type ◽  
Intracellular Growth ◽  
Cell Growth Arrest ◽  
Transgenic Parasites

ABSTRACT Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B3. A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

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.

scholarly journals Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance inLeishmania major

2011 ◽  
Vol 55 (3) ◽  
pp. 1045-1052 ◽  
Author(s):  
José Ignacio Manzano ◽  
Luis Carvalho ◽  
José M. Pérez-Victoria ◽  
Santiago Castanys ◽  
Francisco Gamarro
Keyword(s):  
Leishmania Major ◽  
Alternative Therapy ◽  
Atp Synthesis ◽  
Drug Efflux ◽  
Wild Type ◽  
Mechanisms Of Resistance ◽  
Glycolytic Atp ◽  
Drug Free

ABSTRACTTafenoquine (TFQ), an 8-aminoquinoline used to treat and preventPlasmodiuminfections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity bothin vitroandin vivo, where it targetsLeishmaniamitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood thatLeishmaniawill develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistantLeishmania majorpromastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in allLeishmanialines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance inLeishmania.

Interaction of Stigmasterol with Trypanosomal Uridylyl Transferase, Farnesyl Diphosphate Synthase and Sterol 14α-demethylase: An In Silico Prediction of Mechanism of Action

2019 ◽  
Vol 16 (7) ◽  
pp. 799-807
Author(s):  
Mohammed Auwal Ibrahim ◽  
Murtala Bindawa Isah ◽  
Nasir Tajuddeen ◽  
Saadatu Auwal Hamza ◽  
Aminu Mohammed
Keyword(s):  
Drug Targets ◽  
Neglected Tropical Diseases ◽  
Binding Free Energy ◽  
Economic Loss ◽  
Farnesyl Diphosphate ◽  
New Drugs ◽  
Farnesyl Diphosphate Synthase ◽  
Highly Hydrophobic

Background: Trypanosomiasis is one of the neglected tropical diseases and continues to cause serious morbidity, mortality and economic loss. Current anti-trypanosomal drugs are antiquated and suffer from a number of serious setbacks, thereby necessitating the search for new drugs. Stigmasterol has previously demonstrated in vitro and in vivo anti-trypanosomal activity. Methods: Herein, stigmasterol was docked into three validated anti-trypanosomal drug targets; uridylyl transferase, farnesyl diphosphate synthase and sterol 14α-demethylase, in order to elucidate the possible biochemical targets for the observed anti-trypanosomal activity. Results: The binding free energy between stigmasterol and the enzymes was in the order; sterol 14α-demethylase (-8.9 kcal/mol) < uridylyl transferase (-7.9 kcal/mol) < farnesyl diphosphate synthase (-5.7 kcal/mol). At the lowest energy docked pose, stigmasterol interacts with the active site of the three trypanosomal enzymes via non-covalent interactions (apart from hydrogen bond) while highly hydrophobic stigmasterol carbon atoms (21 and 27) were crucial in the interaction with varying residues of the three anti-trypanosomal targets. Conclusion: Therefore, results from this study might suggest that stigmasterol mediated the antitrypanosomal activity through interaction with the three anti-trypanosomal targets but with more preference towards sterol 14α-demethylase.

scholarly journals Tn5 Transposase with an Altered Specificity for Transposon Ends

2002 ◽  
Vol 184 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Todd A. Naumann ◽  
William S. Reznikoff
Keyword(s):  
Specific Binding ◽  
Random Mutagenesis ◽  
Initial Step ◽  
Wild Type ◽  
Dam Methylase ◽  
End Sequences ◽  
Tn5 Transposase ◽  
Bacterial Transposon

ABSTRACT Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE). In Escherichia coli, IE is methylated by Dam methylase (IEME). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IEME-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IEME defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.

scholarly journals Transient Down-Regulation of Nucleoside Transporter 3 Gene Expression as a Drug Target in Leishmania major Using Antisense RNA Technology

2019 ◽  
Author(s):  
Farideh TOHIDI ◽  
Bahram KAZEMI ◽  
Mojgan BANDEHPOUR ◽  
Iraj SHARIFI ◽  
Mohammad Reza RABIEI ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antisense Rna ◽  
Type Strain ◽  
Wild Type Strain ◽  
Leishmania Major ◽  
Leishmania Infection ◽  
Nucleoside Transporter ◽  
Wild Type

Background: This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite. Methods: Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions. Results: Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites. Conclusion: The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major. More studies are required to obtain a new approach for treating Leishmania infection.

scholarly journals Binding of Calmodulin to the Carboxy-Terminal Region of p21 Induces Nuclear Accumulation via Inhibition of Protein Kinase C-Mediated Phosphorylation of Ser153

2005 ◽  
Vol 25 (16) ◽  
pp. 7364-7374 ◽  
Author(s):  
Aina Rodríguez-Vilarrupla ◽  
Montserrat Jaumot ◽  
Neus Abella ◽  
Núria Canela ◽  
Sonia Brun ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Carboxy Terminal

ABSTRACT Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

scholarly journals Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization

2003 ◽  
Vol 198 (11) ◽  
pp. 1689-1698 ◽  
Author(s):  
Ben L. Kelly ◽  
Daniel B. Stetson ◽  
Richard M. Locksley
Keyword(s):  
Drug Target ◽  
Leishmania Major ◽  
Genomic Organization ◽  
Vaccine Candidate ◽  
Single Copy ◽  
Wild Type ◽  
Immunodeficient Mice ◽  
Human Leishmaniasis ◽  
Parasite Viability

The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell–deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack-null parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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