Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii [Metabolism]

October 30th, 2015 by Nitzsche, R., Zagoriy, V., Lucius, R., Gupta, N.

Toxoplasma gondii is a widespread protozoan parasite, infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here, we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the TCA cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. Growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine, but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.
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Alternative Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and can Activate Cyclin-Dependent Kinase in Xenopus Oocytes [Molecular Bases of Disease]

October 30th, 2015 by

KV10.1 is a voltage-gated potassium channel expressed selectively in mammalian brain, but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lack the transmembrane domains of the channel, and would produce cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitate with the full-length channel and induce a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect is mechanistically different. E65 requires a tetramerization domain and induces a reduction in the overall expression of full-length KV10.1, while E70 mainly affects its glycosylation pattern. E65 triggers activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of non-canonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy.
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MiR-346 Up-regulates Argonaute 2 (AGO2) Protein Expression to Augment the Activity of Other MiRNAs and Contributes to Cervical Cancer Cell Malignancy [Cell Biology]

October 30th, 2015 by Guo, J., Lv, J., Liu, M., Tang, H.

microRNAs (miRNAs) are a class of post-transcriptional regulators of gene expression, and AGO2 is essential for miRNA activity. In this study, we focused on the regulation of AGO2 by miR-346 and the consequences in cervical cancer cells. miR-346 enhanced the expression of AGO2, resulting in the increased activity of other miRNAs and contributing to the malignancy of HeLa cells. GRSF1 participated in the regulation of AGO2 by miR-346, and the middle sequence of miR-346 was vital for synergy effect of miR-346 and GRSF1. We determined that miR-346 promoted the migration and invasion of HeLa cells. In summary, we are the first to report that AGO2 is positively regulated by miRNA and that GRSF1 participates in the miRNA pathway.
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Kruppel homolog 1 inhibits insect metamorphosis via direct transcriptional repression of Broad-complex, a pupal specifier gene [Signal Transduction]

October 30th, 2015 by

The Broad-complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone, 20E), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Kruppel homolog 1 gene (Kr-h1) has been known as a JH-early-inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans-elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter, and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.
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Non-equivalence of key positively charged residues of the free fatty acid 2 receptor in the recognition and function of agonist versus antagonist ligands [Protein Structure and Folding]

October 29th, 2015 by

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled Free Fatty Acid 2 (FFA2) receptor and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabelled FFA2 antagonist in order to probe ligand binding to FFA2 and in combination with mutagenesis and molecular modelling studies define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, while agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.
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Structural and empirical analyses define side-chain clash repair as the key mediator of stability & affinity improvements in an optimized biotherapeutic scFv [Molecular Biophysics]

October 29th, 2015 by

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins which should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using CDR-restricted mutagenesis and tailored selection and screening strategies, and carries 4 mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants were generated to interrogate the individual contribution of each of the 4 mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.
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Proteolytic cleavage driven by glycosylation [Glycobiology and Extracellular Matrices]

October 29th, 2015 by Kotzler, M. P., Withers, S. G.

Proteolytic processing of human Host Cell Factor 1 (HCF-1) to its mature form, was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc Transferase (OGT) (Science 342, 1235-1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed.

Commensal bacteria-induced interleukin-1{beta} (IL-1{beta}) secreted by macrophages upregulates hepcidin expression in hepatocytes by activating the bone morphogenetic protein signaling pathway [Metabolism]

October 29th, 2015 by Shanmugam, N. K. N., Chen, K., Cherayil, B. J.

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that upregulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here, we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria upregulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes, and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in the hepatocytes and in mouse liver as indicated by increased phosphorylation of small-mothers against decapentaplegic (SMAD) proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling, or with a neutralizing antibody to BMP2, prevented IL-1β-induced upregulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression, and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β upregulates hepcidin.
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ATP-binding Cassette Sub-family C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs [Metabolism]

October 29th, 2015 by

The ubiquitous efflux transporter ATP-binding cassette sub-family C member 5 (ABCC5) is present at high levels in the blood-brain barrier, neurons and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens we show that Abcc5-/- mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate (NAAG) was 2.4-fold higher in Abcc5-/- brain. The metabolites that accumulated in Abcc5-/- tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay ABCC5 transported the metabolites detected in our screen and a wide range of peptides containing a C-terminal glutamate, but not glutamate and aspartate themselves. ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid and N-methyl-D-aspartate (NMDA); the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2-asphyxiation several immediate early genes were expressed at lower levels in Abcc5-/- brains than in wildtype brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins and drugs.

Conformational Changes in the Endosomal Sorting Complex Required for Transport-III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation [Enzymology]

October 29th, 2015 by Tan, J., Davies, B. A., Payne, J. A., Benson, L. M., Katzmann, D. J.

Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal Sorting Complex Required for Transport-III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a MIT-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here, we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction in concert with a more open conformation of the Ist1 core resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.
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