The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis

The galactolipids, mono- and digalactosyldiacylglycerol (DGDG), are the most common nonphosphorous lipids in the biosphere and account for 80% of the membrane lipids found in green plant tissues. These lipids are major constituents of photosynthetic membranes (thylakoids), and a large body of evidence suggests that galactolipids are associated primarily with plastid membranes in seed plants. A null-mutant of Arabidopsis (dgd1), which lacks the DGDG synthase (DGD1) resulting in a 90% reduction in the amount of DGDG under normal growth conditions, accumulated DGDG after phosphate deprivation up to 60% of the amount present in the wild type. This observation suggests the existence of a DGD1-independent pathway of galactolipid biosynthesis. The fatty acid composition of the newly formed DGDG was distinct, showing an enrichment of 16-carbon fatty acids in the C-1 position of the glycerol backbone of DGDG. Roots with their rudimentary plastids accumulated large amounts of DGDG after phosphate deprivation, suggesting that this galactolipid may be located in extraplastidic membranes. Corroborating evidence for this hypothesis was obtained directly by fractionation of subcellular membranes from leaf tissue and indirectly by lipid analysis of the phosphate-deprived fad3 mutant primarily deficient in extraplastidic fatty acid desaturation. The discovery of extraplastidic DGDG biosynthesis induced by phosphate deprivation has revealed a biochemical mechanism for plants to conserve phosphate. Apparently, plants replace phospholipids with nonphosphorous galactolipids if environmental conditions such as phosphate deprivation require this for survival.

The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH1

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg−1 chlorophyll h−1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.

A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.

Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.

Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.

An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for l-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice

Macrophages are considered to be the mediators of resistance to extra-intestinal Salmonella infections. Nevertheless, the initial cellular response to Salmonella infections consists primarily of polymorphonuclear leukocytes (PMN). To determine whether PMN serve an important function for the infected host, we made mice neutropenic with the rat mAb to RB6–8C5 and infected them i.v. with ≈103 Salmonella dublin or an isogenic derivative that lacks the virulence plasmid (LD842). We infected BALB/c mice, which have a point mutation in the macrophage-expressed gene Nramp1 that makes them susceptible to Salmonella, and BALB/c.D2 congenic mice, which have the wild-type Nramp1 gene that makes them resistant to Salmonella. Both mouse strains were resistant to LD842, and neutropenia made only the BALB/c strain susceptible to this infection. Neutropenic congenic mice, however, were susceptible only to wild-type S. dublin (plasmid+). These results show a complex interplay between plasmid-virulence genes in Salmonella, host macrophages, and PMN. Mice with normal macrophages need PMN to defend against nontyphoid Salmonella that carry a virulence plasmid but not against Salmonella without virulence plasmids. Mice with a mutant Nramp1 gene need PMN to defend against all Salmonella, even those that lack virulence plasmids. These results, plus the evidence that PMN kill Salmonella efficiently in vitro, suggest that Salmonella have adapted to grow inside macrophages where they are relatively sheltered from PMN. The adaptations that allow Salmonella to survive in macrophages do not protect them from PMN.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsα

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (Gsα) to stimulate enzyme activity. Concentration–response curves with Gsα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572–F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042–T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042–T1051) in the C2 domain of the enzyme to modulate Gsα-elicited stimulation of activity.

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.