Evidence for dual binding sites for DDT in Insect Sodium Channels [Computational Biology]

December 4th, 2015 by Du, Y., Nomura, Y., Zhorov, B. S., Dong, K.

DDT, the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both PyRs remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, while two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed ten previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.

Regulation of sphingolipid biosynthesis by the morphogenesis checkpoint kinase Swe1 [Enzymology]

December 3rd, 2015 by

Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1 regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle.

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Eschericha coli [Microbiology]

December 3rd, 2015 by Pannen, D., Fabisch, M., Gausling, L., Schnetz, K.

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (=EcpR) and with DctR. Our data suggest that the MatA dependent transcription regulation is mediated by the MatA-RcsB heterodimer, and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet that is coordinating phosphorylation, of conserved residues, as well as of surface exposed residues for activity of RcsB homo- and heterodimers. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various auxiliary regulators and their differential dependence on phosphorylation thus adds an additional level of control to the Rcs system that is operating at the output level.

Biochemical and structural characterization of the interaction between the Siderocalin NGAL/LCN2 and the N-terminal domain of its endocytic receptor SLC22A17. [Cell Biology]

December 3rd, 2015 by

The neutrophil gelatinase associated lipocalin (NGAL, aslo known as LCN2) and its cellular receptor (LCN2-R) are involved in many physiological and pathological processes such as cell differentiation, apoptosis and inflammation. These pleiotropic functions mainly rely on NGALs siderophore mediated iron transport properties. However the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo-state to form a fuzzy complex. The relatively weak affinity (≈ 10μM) between hLCN2-R-NTD and apoNGAL suggests that the N-terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, hLCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor, or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.
  • Posted in Journal of Biological Chemistry, Publications
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Interaction between a domain of a negative regulator of the RAS-ERK pathway, SPRED1, and the GTPase-Activating Protein-Related Domain of neurofibromin is implicated in Legius Syndrome and Neurofibromatosis Type 1. [Signal Transduction]

December 3rd, 2015 by

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the RAS-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids (aa) and the C-terminal 20 aa of the GTPase-Activating Protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.
  • Posted in Journal of Biological Chemistry, Publications
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The molecular basis of TnrA control by glutamine synthetase in Bacillus subtilis [Microbiology]

December 3rd, 2015 by Hauf, K., Kayumov, A., Gloge, F., Forchhammer, K.

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active, but unable to interact with TnrA. This conformation was stabilized by phosphorylated MSX, fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no more able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. SPR analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size-exclusion and multi-angle light scattering analysis (MALS), the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

Recycling and Endosomal Sorting of Protease-activated Receptor-1 is Distinctly Regulated by Rab11A and Rab11B [Signal Transduction]

December 3rd, 2015 by Grimsey, N. J., Coronel, L. J., Cordova, I. C., Trejo, J.

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naive receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study, we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.

Secreted frizzled-related protein 5 diminishes cardiac inflammation and protects the heart from ischemia-reperfusion injury [Signal Transduction]

December 2nd, 2015 by

Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 knock-out (KO) and wild type (WT) mice were subjected to ischemia/reperfusion (I/R). Whereas Sfrp5-KO mice exhibited no detectable phenotype compared to WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression compared to WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.
  • Posted in Journal of Biological Chemistry, Publications
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DndEi exhibits helicase activity essential for DNA phosphorothioate modification and ATPase activity strongly stimulated by DNA substrate with a GAAC/GTTC motif [Microbiology]

December 2nd, 2015 by

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared to canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as an ATP-dependent DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e., the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strands separation and intense ATP hydrolysis.
  • Posted in Journal of Biological Chemistry, Publications
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Interaction between RING1 (R1) and Ubiquitin-Like (UBL) Domain Is Critical for the Regulation of Parkin Activity [Neurobiology]

December 2nd, 2015 by Ham, S. J., Lee, S. Y., Song, S., Chung, J.-R., Choi, S., Chung, J.

Parkin is an E3 ligase that contains an UBL domain in the N-terminus and an R1-in-between-ring (IBR)-RING2 (R2) motif in the C-terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at S65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at S65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on the interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, is also suppressed by competitive binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations-associated with Parkinson′s disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by CCCP treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1, and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.