Mammalian Forms Research Articles

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

Although cytochrome P450cam from Pseudomonas putida, the archetype for all heme monooxygenases, has long been known to have a closed active site, recent reports show that the enzyme can also be crystallized in at least two clusters of open conformations. This suggests that the enzyme may undergo significant conformational changes during substrate binding and catalytic turnover. However, these conformations were observed in the crystalline state, and information is needed about the conformations that are populated in solution. In this study, double electron-electron resonance experiments were performed to observe substrate-induced changes in distance as measured by the dipolar coupling between spin labels introduced onto the surface of the enzyme on opposite sides of the substrate access channel. The double electron-electron resonance data show a decrease of 0.8 nm in the distance between spin labels placed at S48C and S190C upon binding the substrate camphor. A rotamer distribution model based on the crystal structures adequately describes the observed distance distributions. These results demonstrate conclusively that, in the physiologically relevant solution state, the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results in conversion to the closed P450cam-C form. This approach should be useful for investigating many other P450s, including mammalian forms, in which the role of conformational change is of central importance but not well understood.

Discovery and Biochemical Characterization of Plasmodium Thioredoxin Reductase Inhibitors from an Antimalarial Set

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.

Multi-species Analyses of Direct Activators of the Constitutive Androstane Receptor

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.

Cross-sectional study of hepatic CYP1A and CYP3A enzymes in hybrid striped bass, channel catfish and Nile tilapia following oxytetracycline treatment

Terramycin for Fish® (oxytetracycline, OTC) is one of three approved drugs for therapeutic treatment of fish in the United States. Nothing is known, however, of the effects of this therapeutic on drug metabolizing enzymes in fish post-treatment. The main purpose of the study was to examine whether the fish CYP1A and CYP3A enzymes would cross-react with antibodies to known mammalian cytochrome P-450 forms (CYP1A1 and CYP3A). Observational feeding studies of OTC effects were conducted in hybrid striped bass, channel catfish and Nile tilapia. Oxytetracycline was mixed into the feed to achieve a daily dose of 82.8mg per kg body weight at a feeding rate of 1% body weight per day. Hepatic microsomes of each fish were prepared and Western blotting of CYP1A1 and CYP3A4 and enzyme assays of CYP1A2 and CYP3A4 were performed prior to OTC treatment and on post-treatment days 1, 6, 11 and 21. Both goat anti-rat CYP1A1 and rabbit anti-human CYP3A4 showed good cross-reactivity with all three species in this study. All three species exhibited distinct perturbations in one or more of the variables examined on day 1 post-treatment. Immediately following the 10-day medication period, relative liver weight (RLW) of hybrid striped bass was increased 44% and remained elevated through post-treatment day 21. Increased CYP3A4 enzyme activity and protein abundance were noted in channel catfish and Nile tilapia, respectively. This observational approach demonstrated species differences both in control activities and in the timing and extent of hepatic responses to OTC. The unique perturbations of hepatic CYP450 enzymes in different fish species to OTC treatment observed in this study may have relevance for the use of additional antibiotics or other therapeutics used in aquaculture.

A poxviral homolog of the Pellino protein inhibits Toll and Toll-like receptor signalling

Toll-like receptor (TLR) signalling pathways constitute an evolutionarily conserved component of the host immune response to pathogenic infection. Here, we describe the ability of a virally encoded form of the Pellino protein to inhibit Toll- and TLR-mediated activation of downstream Rel family transcription factors. In addition to inhibiting drosomycin promoter activation by Spatzle in Drosophila melanogaster cells, viral Pellino attenuates the activation of NF-κB by TLR signalling components and by the TLR4 ligand, LPS, in human cells. We propose that viral Pellino, like mammalian Pellinos, contains a forkhead-associated domain but differs from the mammalian forms in that it lacks a complete and functional RING-like domain. We produce a homology model and present experimental data to support this model by demonstrating that, like mammalian Pellinos, viral Pellino can interact with IRAK-1 via its forkhead-associated domain, whereas unlike its mammalian counterparts, it fails to post-translationally modify IRAK-1. Furthermore, we demonstrate that viral Pellino can functionally antagonise the activity of human Pellino3S. Thus, our findings identify potential immunoevasive capabilities possessed by a poxviral homolog of the Pellino protein and add growing evidence for a likely role for Pellino proteins in Toll and TLR signalling.

Structural architecture ofGaldieria sulphurariaDCN1L

NEDD8ylation is a major regulatory event for cullin-RING E3 ubiquitin ligases (CRL).1,2 NEDD8ylation activates the ubiquitylation activity of CRLs,3–5 which serves to regulate a wide variety of cellular processes6 by selecting specific targets for destruction in the proteasome.7 NEDD8 E3 ligases catalyze ligation of the ubiquitin-like protein NEDD8 (Rub1 in yeast) to the C-terminal winged-helix domain of the CRL “cullin” subunit.8 NEDD8ylation increases the ubiquitin (Ub) ligase activity of CRLs by enhancing binding of the CRL RING subunit to Ub-charged Ub-conjugating (E2) enzymes,9 and by driving conformational changes that permit the RING subunit to productively orient Ub-E2 conjugates with bound CRL substrates for catalysis.10 However, it is unclear how the NEDD8 E3 ligase catalyses its reaction, what its exact composition is, and how it is regulated. Moreover, current structural and functional models of the NEDD8 E3 ligase are based on the Saccharomyces cerevisiae system,8 in which, an integral NEDD8 E3 ligase subunit, DCN1, has low sequence identity to mammalian DCN1-like proteins, and several amino acid insertions, which together may impede understanding of NEDD8 E3 function in a mammalian system. Protein NEDD8ylation is analogous to ubiquitylation.11 NEDD8-activating enzymes (E1), conjugating enzymes (E2), and ligases (E3) are required for the ligation of NEDD8 to its target CRL(s).2 Each enzyme catalyzes transfer of NEDD8 to its respective acceptor protein through NEDD8’s C-terminal glycine carboxylate. First, in an ATP dependent reaction, NEDD8 is covalently linked to the active site cysteine of a heterodimeric E1 enzyme.12 NEDD8 is then transferred to the active site cysteine of an E2 enzyme (Ubc12).13 The E3 enzyme complex then serves to ensure reaction specificity and to promote chemical catalysis en route to NEDD8 transfer to a specific cullin lysine.8 The NEDD8 enzyme complex contains the cullin substrate, a RING domain containing subunit with bound NEDD8-E2 conjugate, and a DCN1-like protein (DCN1L).8 Mammalian cells utilize five DCN1L paralogs,14 in contrast to only two cullin-associated RING paralogs.8 The exact function of each DCN1L is currently unknown. Some DCN1Ls may work in a non-redundant manner as exemplified by human DCN1L3, which preferably interacts with human cullin 3 (Cul3), and localizes Cul3 to the plasma membrane.14 DCN1Ls generally contain two domains, an N-terminal domain that is unique for each paralog, and a conserved C-terminal domain, termed the PONY-domain.15,16 The purpose of the N-terminal domain is not well understood, but likely aids in reaction specificity and cellular localization.14 The PONY-domain, however, is integral to protein NEDD8ylation, and is functional in CRL neddylation in the absence of the N-terminal domain.8,16 Recently, Scott et al. solved the structure of yeast DCN1 in complex with the winged-helix domain of the S. cerevisiae cullin, Cdc53, and provided some clarification for the composition and mechanism of NEDD8 E3s.8 They identified DCN1 and the S. cerevisiae CRL RING subunit, Hrt1, as the major functional components of the yeast NEDD8 E3 ligase. Cdc53 appears to serve as the scaffold for its own NEDD8ylation, as it is already binds Hrt1 in the canonical CRL complex and binds tightly to DCN1.8 Hrt1 binds the NEDD8-charged E2, Ubc12, and enhances chemical catalysis.8 DCN1 facilitates catalysis, presumably by optimizing the orientation of the NEDD8-Ubc12 conjugate with the reactive Cdc53 lysine residue.8 Here, we report the structure of a DCN1L from Galdieria sulphuraria (Gs-DCN1L) to 1.3 A resolution. As all DCN1L structures deposited in the PDB to date are from S. cereviseae DCN1 (Sc-DCN1), which has low sequence identity and frequent amino acid insertions within its polypeptide chain as compared to mammalian DCN1Ls, we sought a DCN1L that would be more comparable with mammalian forms. Gs-DCN1L was identified in genomic sequences17 and expressed sequence tag data from G. sulphuraria.18 Gs-DCN1L was targeted for structural characterization when crystallization trials for the homologous protein, Mus musculus Dcn1D1 (Mm-Dcn1D1), failed to yield crystals. Gs-DCN1L shares higher sequence identity with mammalian DCN1Ls than Sc-DCN1, with a low frequency of amino acid insertions and gaps. Gs-DCN1L shares 25% amino acid identity with Sc-DCN1. At the cullin-binding region Gs-DCN1L and Sc-DCN1 are very similar in fold and in sequence, indicative of a conserved cullin-binding mode. Gs-DCN1L and Sc-DCN1 structures diverge markedly near the cullin interaction surface. In this region Gs-DCN1L has a disordered loop that exposes a hydrophobic pocket, whereas, the comparable polypeptide segment of Sc-DCN1 completely covers the analogous hydrophobic pocket, and is well ordered.8,15,16 Sequence analysis indicates that this intriguing structural divergence is conserved among Gs-DCN1L and the mammalian DCN1L paralogs and may provide clues for additional DCN1L functionalities.

Protein Keeps Adjacent Tissues Growing in Synchrony

Why are hearts heart-shaped, and kidneys shaped like kidneys? More generally, what keeps multicellular creatures, and their individual parts, correctly proportioned even as they increase a thousand-fold or more in size? Despite the ubiquity of the phenomenon and the intriguing nature of the question, relatively little is known about the molecular mechanisms that regulate proportional growth. In this issue of PLoS Biology, Duarte Mesquita, Andres Dekanty, and Marco Milan offer one clue—identifying a protein that slows growth in one part of a fly wing when growth in another part is reduced. Within the fly larva, cell clusters called imaginal discs develop as the larva grows. These clusters play no role in the larval stage of life; instead, they are the primordia of adult structures, including the wings. During metamorphosis, they reorganize, fuse, and grow to create the adult fly. To examine the effect of growth retardation in one group of cells on the growth rate in another, the authors added the gene for several protein synthesis inhibitors, including the poison ricin, to one section (anterior or posterior) of the wing imaginal disc, leaving the other section untouched. The ricin gene’s expression could be turned off with cold temperatures and on with warm ones, and the larvae were initially grown in a cool room to allow normal wing disc development, then switched to a warm one, stunting growth. They found that reducing growth in one section caused a similar reduction in the other section. When growth in the treated section was reduced more, by exposing the larvae to warm temperatures for longer, growth in the untreated section fell in lockstep. The result in all cases was a smaller, but still well-proportioned wing. There was no impact on other organs, or on the overall size of the fly. The reduction in growth in the treated region (a so-called autonomous effect) was not surprising, since reducing protein synthesis reduces cell growth and proliferation. To explain the reduction in the neighboring region, a so-called non-autonomous effect, the authors first showed it was due mainly to a reduced number of cells, rather than a reduction in cell size, indicating an effect on mitosis or cell death. They next examined the role of the protein dp53, a transcription factor and tumor suppressor known to regulate the cell cycle and apoptosis in times of stress. Reducing dp53 in the treated region had little effect on the final size of that section of the wing, consistent with the more profound negative effect of protein depletion on growth. But reducing dp53 in the treated region reversed the normally inhibited growth in the untreated region—when the activity or the quantity of the protein was reduced, the untreated wing region developed normally, despite the smaller size of the adjacent, treated, region, indicating a central role for dp53 in communicating between them. dp53 acted both directly on the adjacent tissue to reduce cell growth, and indirectly, through proteins called caspases, to reduce cell proliferation. The results shed light on one mechanism for insuring development of a wellproportioned organ in a multicellular creature, and it is likely that p53, the mammalian form of dp53, plays a similar role in humans in the regulation of growth in closely adjacent tissues. Many mysteries remain, however. Coordinated growth over longer distances, such as between two limbs, will almost certainly involve multiple other mechanisms, and it will be interesting to see if p53 serves as a local enforcer of longer-range signals exchanged between more distant paired organs.

The four trypanosomatid eIF4E homologues fall into two separate groups, with distinct features in primary sequence and biological properties

Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.

Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications

Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.

A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens

RNA editing regulates mitochondrial gene expression in trypanosomatid pathogens by creating functional mRNAs. It is catalyzed by a multi-protein complex (the editosome), and is found to be essential in both insect stage and mammalian blood stream form of Trypanosoma brucei. This particular form of RNA editing is unique to trypanosomatids, and thus provides a suitable drug target in trypanosomatid pathogens. Here, we demonstrate the feasibility of a rapid and sensitive fluorescence-based reporter assay to monitor RNA editing based on ribozyme activity. We could validate our new assay using previously identified inhibitors against the essential RNA editing ligase. The principle advantages of this assay are: (i) the use of non-radioactively labeled materials, (ii) sensitivity afforded by fluorescence instrumentation applicable to high-throughput screening of chemical inhibitors against the essential editosome and (iii) a rapid and convenient ‘mix and measure’ type of assay in low volume with a high signal to noise ratio. This assay should enhance rapid identification and characterization of the editosome inhibitors primarily based on the overall composition of the editosomes from T. brucei. These inhibitors could also be tested against the editosomes from the closely related pathogens including T. cruzi and Leishmania species.

Calflagin Inhibition Prolongs Host Survival and Suppresses Parasitemia in Trypanosoma brucei Infection

African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.

Abstract 826: Expression levels of circulating soluble-EphA2 receptor in cancer patients

Abstract EphA2 is a receptor tyrosine kinase that critically controls many aspects of cell behavior. It is expressed in a manner that is consistent with the regulation of cell growth, migration and invasion. Analysis by IHC has shown that elevated expression of EphA2 exhibits predictive value in lung cancer survival and recurrence. Importantly, current data suggests that targeting EphA2 could be beneficial and is currently being tested in an ovarian cancer setting. We developed a prototype ELISA for circulating EphA2 receptor in order to investigate expression levels of the soluble form of EphA2 in certain cancer types. We have developed a prototype ELISA which measures circulating human EphA2 receptor. 96 well Nunc plates were coated with an affinity-purified goat polyclonal antibody specific for the extracellular domain (ECD) of human EphA2. Human serum and plasma samples were diluted 5-fold in a sample diluent with blocking reagents. The standard was a mammalian cell-derived form of the ECD for the human EphA2 receptor. Standards and samples were incubated on the plates for 2 Hours, 37C. The detector antibody was a biotinylated affinity-purified goat polyclonal antibody and was added to the plates for 1 Hour, 37C. Plates were washed and a streptavidin-HRP conjugate was added to the plates for 30 minutes, 22C. A final wash was performed and a TMB substrate was added to the plates for color development. The reaction was stopped with sulfuric acid and plates were read at an absorbance of 450nm. Data generated to date using this prototype ELISA indicates that over-expression of soluble-EphA2 occurs in many cancers versus normal samples tested. The 95% CI of total normal plasma and serum was 672_1306 and 894_1429pg/mL, respectively. Capture Inhibition studies revealed specificity of the assay in human samples (100% inhibition). Parallelism studies revealed acceptable performance in serially diluted samples. Significant over-expression of soluble-EphA2 was observed in benign lung cancers versus normals (100%, 10/10) while staged lung cancers exhibited significantly lower levels of circulating EphA2 (p<0.001) compared to benign (96%, 148/154). Elevations of circulating EphA2 were found in glioblastoma (73%, 22/30), ovarian (48%, 28/59), pancreatic (46%, 19/41), and late stage breast (44%, 15/34) cancer patients. Immunoprecipitation experiments are ongoing to visualize soluble EphA2 by western blot, with initial data showing a specific band at ∼60kD. This study describes the discovery of circulating EphA2 receptor in human samples using a prototype ELISA. Levels of soluble-EphA2 are over-expressed in glioblastoma, ovarian, pancreatic, and late stage breast cancer samples. Decreased expression of the circulating form of EphA2 is observed in staged lung cancer versus benign. Measuring the levels of the soluble-EphA2 in circulation may have potential utility for cancer patients who may benefit from EphA2 targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 826.

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani.

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a approximately 33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.

Synthetic atpenin analogs: Potent mitochondrial inhibitors of mammalian and fungal succinate-ubiquinone oxidoreductase

Atpenins and harzianopyridone represent a unique class of penta-substituted pyridine-based natural products that are potent inhibitors of complex II (succinate-ubiquinone oxidoreductase) in the mitochondrial respiratory chain. These compounds block electron transfer in oxidative phosphorylation by inhibiting oxidation of succinate to fumarate and the coupled reduction of ubiquinone to ubiquinol. From our investigations of complex II inhibitors as potential agricultural fungicides, we report here on the synthesis and complex II inhibition for a series of synthetic atpenin analogs against both mammalian and fungal forms of the enzyme. Synthetic atpenin 2e provided optimum mammalian and fungal inhibition with slightly higher potency than natural occurring atpenin A5.

Is it human? identifiers that distinguish animal skeletal remains from human

Even in the most experienced hands it is not always possible to distinguish human from animal skeletal remains. While the skull and vertebrae of most animals possess features quite different to humans, given the common mammalian form it is often difficult to distinguish post-cranial remains, particularly when the articular surfaces are missing. This part of the workshop will present some principles useful for identifying animal skeletal remains and provide examples of animal bones that are typically mistaken for human.

Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3' untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3' UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3' UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5' end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5' UTR, since a hairpin structure abolishes expression of a fused reporter gene.

Gene structure of the Kiss1 receptor-2 ( Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes

Kisspeptin and its receptor, Kiss1r, play an essential role in the control of the onset of puberty in vertebrates. We characterized the cDNA and genomic DNA encoding Kiss1r in Atlantic halibut ( Hippoglossus hippoglossus). The 1146 bp open reading frame predicts a 381 amino acid protein with high homology to the Kiss1r-2 of other teleost fish. Phylogenetic analysis of Kiss1r sequences suggests that the mammalian Kiss1r-1 form arose by way of a gene duplication prior to the emergence of amphibians. Synteny analysis demonstrated the highly conserved nature of the Kiss1r-2 region in teleosts, suggesting that flanking regulatory sequences are also likely to be conserved. Bioinformatic analysis identified six conserved regions in piscine Kiss1r-2 upstream sequences, providing potential targets for future in-depth investigation of Kiss1r-2 regulation. Kiss1r-2 expression in the brain increased coinciding with the onset of puberty. Expression levels in the gonads were two orders of magnitude lower than those of the brain, a characteristic apparently conserved in other fishes, and expression in gonads was only detected in immature fish.

Chemistry and Biology of Galactofuranose‐Containing Polysaccharides

The thermodynamically less stable form of galactose-galactofuranose (Galf)-is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf-containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in-depth understanding of the mechanism of UDP-galactopyranose mutase (UGM), the enzyme that produces UDP-Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either alpha- or beta-Galf residues. UDP-Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi-milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.

Heme Regulation of Human Cystathionine β-Synthase Activity: Insights from Fluorescence and Raman Spectroscopy

Cystathionine beta-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C horizontal lineN bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.