Exploiting Differences in Heme Biosynthesis between Bacterial Species to Screen for Novel Antimicrobials.

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an Escherichia coli YFP strain were replaced with those for the CPD pathway from Staphylococcus aureus (SA) through a sliding modular gene replacement recombineering strategy to generate the E. coli strain Sa-CPD-YFP. Potential inhibitors that differentially target the pathways were identified by screening compound libraries against the YFP-producing Sa-CPD-YFP strain in comparison to a CFP-producing E. coli strain. Using a mixed strain assay, inhibitors targeting either the CPD or PPD heme pathways were identified through a decrease in one fluorescent signal but not the other. An initial screen identified both azole and prodigiosin-derived compounds that were shown to specifically target the CPD pathway and which led to the accumulation of coproheme, indicating that the main target of inhibition would appear to be the coproheme decarboxylase (ChdC) enzyme. In silico modeling highlighted that these inhibitors are able to bind within the active site of ChdC.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

Ferritin in serum and urine: A pilot study.

Serum ferritin reflects total body iron stores, thus a low serum ferritin is used as a parameter of iron deficiency. In healthy adults in Japan, urine ferritin levels were about 5% of serum ferritin levels, with a correlation coefficient of 0.79. It is not known whether a low urine ferritin could serve as a non-invasive screen for iron deficiency. If so, this might be useful for neonates and young children, avoiding phlebotomy to screen for iron deficiency. However, for urinary ferritin screening to be feasible, ferritin must be measurable in the urine and correlate with serum ferritin. Testing should also clarify whether the iron content of ferritin in serum and urine are similar. In this pilot feasibility study we measured ferritin in paired serum and urine samples of healthy adult males, healthy term neonates, growing preterm neonates, and children who had very high serum ferritin levels from liver disorders or iron overload. We detected ferritin in every urine sample, and found a correlation with paired serum ferritin (Spearman correlation coefficient 0.78 of log10-transformed values). These findings suggest merit in further studying urinary ferritin in select populations, as a potential non-invasive screen to assess iron stores.

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis.

During erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate-dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

Two novel mutations in TMPRSS6 associated with iron-refractory iron deficiency anemia in a mother and child.

In an iron deficient child, oral iron repeatedly failed to improve the condition. Whole exome sequencing identified one previously reported plus two novel mutation in the TMPRSS6 gene, with no mutations in other iron-associated genes. We propose that these mutations result in a novel variety of iron-refractory iron deficiency anemia.

Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor α and ß Chain mRNAs.

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3'-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and ß chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRß sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells.

Alternative preprocessing of RNA-Sequencing data in The Cancer Genome Atlas leads to improved analysis results.

MOTIVATION: The Cancer Genome Atlas (TCGA) RNA-Sequencing data are used widely for research. TCGA provides 'Level 3' data, which have been processed using a pipeline specific to that resource. However, we have found using experimentally derived data that this pipeline produces gene-expression values that vary considerably across biological replicates. In addition, some RNA-Sequencing analysis tools require integer-based read counts, which are not provided with the Level 3 data. As an alternative, we have reprocessed the data for 9264 tumor and 741 normal samples across 24 cancer types using the Rsubread package. We have also collated corresponding clinical data for these samples. We provide these data as a community resource. RESULTS: We compared TCGA samples processed using either pipeline and found that the Rsubread pipeline produced fewer zero-expression genes and more consistent expression levels across replicate samples than the TCGA pipeline. Additionally, we used a genomic-signature approach to estimate HER2 (ERBB2) activation status for 662 breast-tumor samples and found that the Rsubread data resulted in stronger predictions of HER2 pathway activity. Finally, we used data from both pipelines to classify 575 lung cancer samples based on histological type. This analysis identified various non-coding RNA that may influence lung-cancer histology. AVAILABILITY AND IMPLEMENTATION: The RNA-Sequencing and clinical data can be downloaded from Gene Expression Omnibus (accession number GSE62944). Scripts and code that were used to process and analyze the data are available from https://github.com/srp33/TCGA_RNASeq_Clinical. CONTACT: stephen_piccolo@byu.edu or andreab@genetics.utah.edu SUPPLEMENTARY INFORMATION: Supplementary material is available at Bioinformatics online.

Structural basis for leucine-rich nuclear export signal recognition by CRM1.

CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 A structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined alpha-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

The Salmonella virulence protein SifA is a G protein antagonist.

Salmonella's success at proliferating intracellularly and causing disease depends on the translocation of a major virulence protein, SifA, into the host cell. SifA recruits membranes enriched in lysosome associated membrane protein 1 (LAMP1) and is needed for growth of Salmonella induced filaments (Sifs) and the Salmonella containing vacuole (SCV). It directly binds a host protein called SKIP (SifA and kinesin interacting protein) which is critical for membrane stability and motor dynamics at the SCV. SifA also contains a WxxxE motif, predictive of G protein mimicry in bacterial effectors, but whether and how it mimics the action of a host G protein is not known. We show that SKIP's pleckstrin homology domain, which directly binds SifA, also binds to the late endosomal GTPase Rab9. Knockdown studies suggest that both SKIP and Rab9 function to maintain peripheral LAMP1 distribution in cells. The Rab9:SKIP interaction is GTP-dependent and is inhibited by SifA binding to the SKIP pleckstrin homology domain, suggesting that SifA may be a Rab9 antagonist. SifA:SKIP binding is significantly tighter than Rab9:SKIP binding and may thus allow SifA to bring SKIP to the SCV via SKIP's Rab9-binding site. Rab9 can measurably reverse SifA-dependent LAMP1 recruitment and the perinuclear location of the SCV in cells. Importantly, binding to SKIP requires SifA residues W197 and E201 of the conserved WxxxE signature sequence, leading to the speculation that bacterial G protein mimicry may result in G protein antagonism.

Multiple active site conformations revealed by distant site mutation in ornithine decarboxylase.

Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle.

X-ray structure determination of Trypanosoma brucei ornithine decarboxylase bound to D-ornithine and to G418: insights into substrate binding and ODC conformational flexibility.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the rate-determining step in the biosynthesis of polyamines. ODC is a proven drug target to treat African sleeping sickness. The x-ray crystal structure of Trypanosoma brucei ODC in complex with d-ornithine (d-Orn), a substrate analog, and G418 (Geneticin), a weak non-competitive inhibitor, was determined to 2.5-A resolution. d-Orn forms a Schiff base with PLP, and the side chain is in a similar position to that observed for putrescine and alpha-difluoromethylornithine in previous T. brucei ODC structures. The d-Orn carboxylate is positioned on the solvent-exposed side of the active site (si face of PLP), and Gly-199, Gly-362, and His-197 are the only residues within 4.2 A of this moiety. This structure confirms predictions that the carboxylate of d-Orn binds on the si face of PLP, and it supports a model in which the carboxyl group of the substrate l-Orn would be buried on the re face of the cofactor in a pocket that includes Phe-397, Tyr-389, Lys-69 (methylene carbons), and Asp-361. Electron density for G418 was observed at the boundary between the two domains within each ODC monomer. A ten-amino acid loop region (392-401) near the 2-fold axis of the dimer interface, which contributes several residues that form the active site, is disordered in this structure. The disordering of residues in the active site provides a potential mechanism for inhibition by G418 and suggests that allosteric inhibition from this site is feasible.

Ornithine decarboxylase promotes catalysis by binding the carboxylate in a buried pocket containing phenylalanine 397.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the decarboxylation of l-Orn to putrescine, a rate-limiting step in the formation of polyamines. The X-ray crystal structures of ODC, complexed to several ligands, support a model where the substrate is oriented with the carboxyl-leaving group buried on the re face of the PLP cofactor. This binding site is composed of hydrophobic and electron-rich residues, in which Phe-397 is predicted to form a close contact. Mutation of Phe-397 to Ala reduces the steady-state rate of product formation by 150-fold. Moreover, single turnover analysis demonstrates that the rate of the decarboxylation step is decreased by 2100-fold, causing this step to replace product release as the rate-limiting step in the mutant enzyme. These data support the structural prediction that the carboxyl-leaving group is positioned to interact with Phe-397. Multiwavelength stopped-flow analysis of reaction intermediates suggests that a major product of the reaction with the mutant enzyme is pyridoximine 5'-phosphate (PMP), resulting from incorrect protonation of the decarboxylated intermediate at the C4' position. This finding was confirmed by HPLC analysis of the reaction products, demonstrating that Phe-397 also plays a role in maintaining the integrity of the reaction chemistry. The finding that the carboxylate-leaving group is oriented on the buried side of the PLP cofactor suggests that ODC facilitates decarboxylation by destabilizing the charged substrate carboxyl group in favor of an electrostatically more neutral transition state.

Target validation for drug discovery in parasitic organisms.

Parasite infections affect billions of humans world-wide, yet the current drugs available for the treatment of many parasitic diseases are either inadequate, or compromised by the development of resistance. Validation of a drug target is an important step in the development of new drugs. Target validation encompasses verifying that a target is primarily responsible for the therapeutic activity of a proven drug, or demonstrating the essential nature of a putative drug target in a parasite, and the capacity for selective inhibition of that target in vivo. Selective toxicity may be achieved by taking advantage of unique parasite biology or biochemistry, or by utilizing differences in metabolism or import. The essential nature of a target may be demonstrated by the correlation of the chemical or genetic reduction of target activity with the loss of parasite growth or virulence. Rescue experiments may demonstrate the single nature of a target. Ultimately, a target must be validated in vivo.