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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Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels [Molecular Biophysics]

October 29th, 2015 by

Calmodulin (CaM), a Ca2+ sensing protein, is constitutively bound to IQ domains of the C-termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca2+-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca2+-independent manner but only the long isoform (hKv7.4a) is regulated by Ca2+/CaM. Ca2+/CaM mediate reduction of the hKv7.4a channel by decreasing channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) which has been linked to progressive hearing loss to further examine the inhibitory effects of Ca2+/CaM on Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that G321S mutation may destabilize CaM binding leading to a decrease in the inhibitory effects of Ca2+ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. The report provides mechanistic insights into critical roles of Ca2+/CaM regulation of Kv7.4 channel in physiological and pathological conditions.

Neutron Crystal Structure of RAS GTPase puts in question the Protonation State of the GTP {gamma}-Phosphate [Enzymology]

October 29th, 2015 by Knihtila, R., Holzapfel, G., Weiss, K., Meilleur, F., Mattos, C.

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.

Muscle wasting in fasting requires activation of NF-{kappa}B and inhibition of AKT/mTOR by the protein acetylase, GCN5 [Molecular Bases of Disease]

October 29th, 2015 by Lee, D., Goldberg, A. L.

NF-κB is best known for its proinflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of NF-kκB's p65 subunit on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα- superrepressor (IκBα-SR) or of p65 mutated at K310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mTORactivities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT-mTOR signaling suggest novel approaches to combat muscle wasting.
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