Asparagine repeat function in a
            <i>Plasmodium falciparum</i>
            protein assessed via a regulatable fluorescent affinity tag

Muralidharan, Vasant; Oksman, Anna; Iwamoto, Mutsuo; Wandless, Thomas J.; Goldberg, Daniel E.

doi:10.1073/pnas.1018449108

Cited by 131 publications

(190 citation statements)

References 24 publications

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“…A variation of this strategy would be to express rapamycin-inducible Di-Cre recipient clones of P. falciparum under early-liver-stage-specific promoters to excise FAS-II genes only during liver-stage development (57). An alternative strategy could utilize regulatable tags, which have successfully been used to demonstrate the essentiality of bloodstage expressed proteins (58,59) but, to date, not for pre-erythrocytic stages.…”

Section: Discussionmentioning

confidence: 99%

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

et al. 2014

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The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

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Section: Discussionmentioning

confidence: 99%

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

et al. 2014

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“…The plasmids thus obtained are pQEPfFHFL, pQE-PfFH∆40 and pQE-PfFH∆120. For 3ʹ-tagging of the endogenous fh gene with GFP in the P. falciparum strain PM1KO (27), the nucleotide fragment corresponding to the fulllength fh gene without the terminator codon was amplified from P. falciparum 3D7 genomic DNA using appropriate oligonucleotides (PfFHpGDBXho1-FP and PfFHpGDB-AvrII-RP; S1 Table) and cloned into the plasmid, pGDB (27) using the restriction sites XhoI and AvrII to yield the plasmid pGDB-PfFH. For recombinant expression of E. coli FumC and FumA enzymes, the nucleotide sequence corresponding to the fulllength genes were PCR amplified using E. coli genomic DNA as template and cloned in pQE30 and pET-DUET (Novagen, Merck), respectively using the restriction sites BamHI and SalI.…”

Section: Generation Of Plasmid Constructs-mentioning

confidence: 99%

“…Though biochemical evidence suggests that FH is mitochondrially localized in P. falciparum (3), microscopic images showing localization to this organelle are not available. To examine the localization of the protein in P. falciparum, the fh gene on chromosome 9 was replaced with DNA encoding FH-RFA (fumarate hydratase-regulatable fluorescent affinity tag that comprises of green fluorescent protein (GFP), E. coli dihydrofolate reductase degradation domain (EcDHFRdd) and a hemagglutinin (HA) tag in tandem) fusion protein by single crossover recombination in PM1KO (27) strain of the parasite (Fig 1a). The genotype of the strain (Fig 1b) was validated by PCR using primers P1-P4 (S1 Table) and used for live-cell imaging after staining with Hoechst and MitoTracker Red CM-H 2 XRos.…”

Section: Mitochondrial Localization Of P Falciparum Fh-mentioning

confidence: 99%

“…O positive erythrocytes from healthy volunteers were added to the culture to a final hematocrit of 2 % for regular maintenance. For examining the localization of PfFH, PM1KO strain (27) was transfected with the plasmid pGDB-PfFH. For this, preloading of erythrocytes (62) with plasmid DNA was carried out by electroporation using a square wave pulse (8 pulses of 365 V each lasting for 1 ms with a gap of 100 ms) program in BioRad-XL electroporator.…”

Section: Complementation Of Fh Deficiency In ∆Fumacb Strain With Pffhmentioning

confidence: 99%

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Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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“…The PCI subunit Rpn6 was found to be an essential component of the 26S proteasome in Saccharomyces cerevisiae (16), Trypanosoma brucei (17), Plasmodium falciparium (18), and Drosophila melanogaster (19). Upon conditional knock-out in S. cerevisiae, only partially assembled complexes lacking all the lid subunits were found, and the cells were arrested in G2/M phase (20).…”

mentioning

confidence: 99%

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Pathare

Nagy

Bohn

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

141

162

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Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.

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Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag

Cited by 131 publications

References 24 publications

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

Biochemical characterization and essentiality of fumarate hydratase

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

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