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.
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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.
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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.
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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.
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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.
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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.

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma [Signal Transduction]

August 18th, 2015 by

The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that miR-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself, but also directly downregulating its two important upstream regulators, namely, ILK and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and, directly and indirectly, repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, miR-542-3p expression level negatively correlated with AKT activity, as well as levels of ILK and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression, and miR-542-3p downregulation contributes to aberrant activation of the AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high-grade astrocytoma.

Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes [Cell Biology]

August 18th, 2015 by Yamaoka, H., Tachibanaki, S., Kawamura, S.

On absorption of light by vertebrate visual pigment, the chromophore, 11-cis retinal, is isomerized to all-trans retinal to activate the phototransduction cascade, which leads to a hyperpolarizing light response. Activated pigment is inactivated by phosphorylation on the protein moiety, opsin. Isomerized all-trans retinal is ultimately released from opsin, and the pigment is regenerated by binding to 11-cis retinal. In this pigment regeneration cycle, the phosphates incorporated should be removed in order that the pigment regains the capability of activating the phototransduction cascade. However, it is not clear yet how pigment dephosphorylation takes place in the regeneration cycle. First in this study, we tried to estimate the dephosphorylation activity in living carp rods and cones, and found that the activity, which is present mainly in the cytoplasm in both rods and cones, is 3-times higher in cones than in rods. Second, we examined at which stage the dephosphorylation takes place; before or after the release of all-trans retinal, during pigment regeneration or after pigment regeneration. For this purpose, we prepared three types of phosphorylated substrates in purified carp rod and cone membranes: phosphorylated bleaching intermediate, phosphorylated opsin, and phosphorylated-and-regenerated pigment. We also examined the effect of pigment regeneration on the dephosphorylation. The results showed that the dephosphorylation does not show substrate preference in the regeneration cycle, and suggested that the dephosphorylation takes place constantly. The results also suggest that, under bright light, some of the regenerated visual pigment remain phosphorylated to reduce the light sensitivity in cones.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease [Plant Biology]

August 17th, 2015 by

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues however significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.

Membrane Curvature-Sensing and -Inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption [Membrane Biology]

August 17th, 2015 by

Islet amyloid polypeptide (IAPP) is a 37-amino-acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for hIAPP than for the less toxic rIAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rIAPP and hIAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.
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