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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Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata [Plant Biology]

October 29th, 2015 by

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pains such as headache, rheumatic pain, toothache and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from active extract of M. alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1 expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice, but not in TRPV1 knockout mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.
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The crystal structure of an integral membrane fatty acid {alpha}-hydroxylase [Lipids]

October 28th, 2015 by

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is comprised of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architectures of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes.

Asn-150 of murine erythroid 5-aminolevulinate synthase modulates the catalytic balance between the rates of the reversible reaction [Protein Structure and Folding]

October 28th, 2015 by Stojanovski, B. M., Ferreira, G. C.

5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. While wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, and thus, ensuring that ALA is predominantly available for heme biosynthesis.
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Hsp90-Cdc37 complexes with protein kinases form cooperatively with multiple distinct interaction sites [Protein Structure and Folding]

October 28th, 2015 by

Protein kinases are the most prominent group of Hsp90 (heat shock protein 90) clients and are recruited to the molecular chaperone by the kinase specific cochaperone Cdc37 (cell division cycle 37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of C. elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the MAP-kinase (mitogen activated protein kinase) Erk2 (extracellular-signal regulated kinase 2) we observe that certain features of the interaction with Cdc37-Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: Evidence for a Common Reaction Mechanism [Protein Structure and Folding]

October 28th, 2015 by Booth, E. S., Basran, J., Lee, M., Handa, S., Raven, E. L.

The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD+. The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine (NFK). This is an O2-dependent process and catalyzed by indoleamine 2,3 dioxygenase (IDO). More than sixty years after these enzymes were first isolated (1), the mechanism of their reaction is not established. We have examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observe formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-Me-L-Trp and a number of other substrate analogues. The data are consistent with a common reaction mechanism for IDO-catalyzed oxidation of tryptophan and other tryptophan analogues.

Insulin dissociates the effects of Liver X Receptor on lipogenesis, endoplasmic reticulum stress and inflammation [Metabolism]

October 28th, 2015 by

Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor Liver X Receptor is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knockdown Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knockout of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation and ER stress, from the negative effects on lipogenesis.

In Vivo Studies in Rhodospirillum rubrum Indicate that Ribulose-1,5,bisphosphate carboxylase/oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways [Microbiology]

October 28th, 2015 by Dey, S., North, J. A., Sriram, J., Evans, B. S., Tabita, F. R.

All organisms possess fundamental metabolic pathways to insure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxy-genase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to insure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knockout mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated representatives of all extant known functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine (MTA) as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic MTA metabolism, suggesting that the active site of Rubisco has evolved to insure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions
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