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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Plasticity in repressor-DNA interactions neutralizes loss of symmetry in bipartite operators [Gene Regulation]

October 28th, 2015 by Jain, D., Narayanan, N., Nair, D. T.

Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. In order to understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators- ORE1, ORX1, ORA1 and ORR3- the AraR-DBD as well as full length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07Å and 1.97Å resolution respectively. These structures are compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with symmetric operator, ORE1 are identical, but their interaction with non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. Chip-Seq data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits.

The Disulfide Bond, but not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu-Zn Superoxide Dismutase (SOD1) Fibrillation [Protein Structure and Folding]

October 28th, 2015 by

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications: Formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact C57-C146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above posttranslational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.
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Hyperosmotic Shock Engages Two Positive Feedback Loops Through Caspase-3 Dependent Proteolysis of JNK1-2 and Bid [Signal Transduction]

October 28th, 2015 by Yue, J., Ben Messaoud, N., Lopez, J. M.

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here we show that JNK1-1 and JNK1-2 are early activated by osmostress and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated JNK1-2 is proteolyzed at Asp385 increasing the release of cytochrome c and caspase-3 activity, and therefore creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and biubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.