Brain Expressed X-Linked 2 Is Pivotal for Hyperactive mTOR-Mediated Tumorigenesis [Signal Transduction]

August 20th, 2015 by

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2-/- MEFs, Pten-/- MEFs, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal-specific Tsc1 knockout mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic-specific Pten knockout mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.

Differential recognition preferences of the three Src homology 3 (SH3) domains from the adaptor CD2-associated protein (CD2AP), and direct association with Ras and Rab interactor 3 (RIN3) [Molecular Biophysics]

August 20th, 2015 by

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three SH3 domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide-array screen based on the recognition motif P-x-P-x-P-R, and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine-nucleotide exchange factor (GEF). CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested, yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry (ITC) data show that the preferred binding motif is P-x-P/A-x-p-R. Two high-resolution crystal structures (1.65 Å and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains to c-CBL, ALIX and RIN3.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Differential recognition preferences of the three Src homology 3 (SH3) domains from the adaptor CD2-associated protein (CD2AP), and direct association with Ras and Rab interactor 3 (RIN3) [Molecular Biophysics]

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport [Lipids]

August 20th, 2015 by

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal (LE/LY) proteins, NPC1 and NPC2. NPC2 is a 16 kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2-/- fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, likely allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of LE/LYs, ultimately effecting cholesterol egress from this compartment.

Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses [Immunology]

August 20th, 2015 by

C-type lectin receptors (CLRs) are an emerging family of pattern-recognition receptors that recognizes pathogens or damaged-tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. Mass spectrometry (MS) analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells (DCs) expressed a set of inflammatory genes in response to cholesterol crystals and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, while other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and activates innate immune responses.

The E3-ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters [Cell Biology]

August 20th, 2015 by

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The E3-ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters [Cell Biology]

The GH130 family of mannoside phosphorylases contains glycoside hydrolases that target beta-1,2 mannosidic linkages in Candida mannan [Glycobiology and Extracellular Matrices]

August 18th, 2015 by

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. beta-mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although a family of glycoside phosphorylases, GH130, have also been shown to target beta-1,2 and beta-1,4 mannosidic linkages. In these phosphorylases bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA03624, which lack the phosphate binding residues are indeed betamannosidases that hydrolyse beta-1,2-mannosidic linkages through an inverting mechanism. As the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerisation of yeast alpha-mannans, it is likely that the two enzymes target the beta-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the -1 and +1 subsites showed a pair of glutamates, Glu227 and Glu268 hydrogen bond to O1 of alpha-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and beta-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for beta-1,2-mannosidic linkages.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The GH130 family of mannoside phosphorylases contains glycoside hydrolases that target beta-1,2 mannosidic linkages in Candida mannan [Glycobiology and Extracellular Matrices]

Site-Directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 on the Physiological Functions of the Rice GTPase-Activating Protein 1 (OsGAP1) [Protein Structure and Folding]

August 18th, 2015 by

The C2 domain is one of the most diverse phospholipid-binding domains mediating cellular signaling. One group of C2-domain proteins are plant-specific and are characterized by their small sizes and simple structures. We have previously reported that a member of this group, OsGAP1, is able to alleviate salt stress and stimulate defense responses, and bind to both phospholipids and an unconventional G-protein, OsYchF1. Here we solved the crystal structure of OsGAP1 to a resolution of 1.63 Å. Using site-directed mutagenesis, we successfully differentiated between the clusters of surface residues that are required for binding to phospholipids versus OsYchF1, which, in turn, is critical for its role in stimulating defense responses. On the other hand, the ability to alleviate salt stress by OsGAP1 is dependent only on its ability to bind OsYchF1 and is independent of its phospholipid-binding activity.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Site-Directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 on the Physiological Functions of the Rice GTPase-Activating Protein 1 (OsGAP1) [Protein Structure and Folding]

The capsule polymerase CslB of Neisseria meningitidis serogroup L catalyzes the synthesis of a complex trimeric repeating unit comprising glycosidic and phosphodiester linkages [Microbiology]

August 18th, 2015 by

Neisseria meningitidis (Nm) is a human pathogen causing bacterial meningitis and sepsis. The capsular polysaccharide (CPS) surrounding Nm is a major virulence factor. The CPS consists of poly-hexosamine-phosphates in NmA and NmX. The capsule polymerases (CPs) of these serogroups are members of the stealth protein family comprising D-hexose-1-phosphate-transferases from bacterial and protozoan pathogens. CslA, one out of two putative CPs of the pathophysiologically less relevant NmL, is one of the smallest known stealth proteins and caught our attention for structure-function-analyses. Since the NmL capsule polymer (CPSL) consists of a trimeric repeating unit ([→3)-β-D-GlcNAc-(1→3)-β-D-GlcNAc-(1→3)-α-D-GlcNAc-(1→OPO3→]n), we speculated that the two predicted CPs (CslA and CslB) work together in the polymer production. Consequently, both enzymes were cloned, overexpressed, and purified as recombinant proteins. Against our expectation, enzymatic testing identified CslB to be sufficient to catalyze the synthesis of the complex trimeric CPSL repeating unit. In contrast, no polymerase activity was detected for CslA, albeit the enzyme facilitated the hydrolysis of UDP-GlcNAc. Bioinformatics analyses identified two glycosyltransferase (GT) domains in CslB. The N-terminal domain modeled with 100% confidence onto a number of GT-A folded proteins while the C-terminal domain modelled with 100% confidence onto TagF, a teichoic acid polymerase from S. epidermidis. Amino acid positions known to have critical catalytic functions in the template proteins were conserved in CslB and their point mutation abolished enzyme activity. CslB represents an enzyme of so far unique complexity regarding both, the catalyzed reaction and enzyme architecture.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The capsule polymerase CslB of Neisseria meningitidis serogroup L catalyzes the synthesis of a complex trimeric repeating unit comprising glycosidic and phosphodiester linkages [Microbiology]

Glutamate Transporter Homolog-Based Model Predicts Anion-{pi} Interaction is the Mechanism for the Voltage-Dependent Response of Prestin [Neurobiology]

August 18th, 2015 by

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions is still unknown. Our three-dimensional model of prestin, using molecular dynamics simulations, predicts that prestin contains eight transmembrane spanning segments, two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Y367, Y486, Y501 and Y508 contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters and motor proteins.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Glutamate Transporter Homolog-Based Model Predicts Anion-{pi} Interaction is the Mechanism for the Voltage-Dependent Response of Prestin [Neurobiology]

Alternative activation mechanisms of Protein Kinase B trigger distinct downstream signaling responses [Signal Transduction]

August 18th, 2015 by

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity towards specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif (HM) are essential for high PKB activity in general. On the other hand we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial HM phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.