Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease.

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

Functional analysis and classification of homozygous and hypomorphic ABCA4 variants associated with Stargardt macular degeneration.

Stargardt macular degeneration (Stargardt disease 1 [STGD1]) is caused by mutations in the gene encoding ABCA4, an ATP-binding cassette protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across photoreceptor membranes. Reduced ABCA4 activity results in retinoid accumulation leading to photoreceptor degeneration. The disease onset and severity vary from severe loss in visual acuity in the first decade to mild visual impairment late in life. We determined the effect of 22 disease-causing missense mutations on the expression and ATPase activity of ABCA4 in the absence and presence of N-Ret-PE. Three classes were identified that correlated with the disease onset in homozygous STGD1 individuals: Class 1 exhibited reduced ABCA4 expression and ATPase activity that was not stimulated by N-Ret-PE; individuals homozygous for these variants had an early disease onset (≤13 years); Class 2 showed reduced ATPase activity with limited stimulation by N-Ret-PE; these correlated with moderate disease onset (14-40 years); and Class 3 displayed high expression and ATPase activity that was strongly activated by N-Ret-PE; these were associated with late disease onset (>40 years). On the basis of our results, we introduce a functionality index for gauging the effect of missense mutations on STGD1 severity. Our studies support the mild phenotype exhibited by the p.Gly863Ala, p.Asn1868Ile, and p.Gly863Ala/p.Asn1868Ile variants.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO4 :Ce3+ ,Tb3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 109 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl11 O19 :Tb3+ ) and BAM (BaMgAl10 O17 :Eu2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y2 O3 :Eu3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc.

Bacteriophage-induced aggregation of oil sands tailings.

Very large quantities of tailings are produced as a result of processing oil sands. After the sand particles settle out, a dense stable mixture of clay, silt, water with residual bitumen, salts, and organics called mature fine tailings (MFT) can remain in suspension for decades. Research into developing methods that would allow consolidation and sedimentation of the suspended particles is ongoing. We have studied the ability of a filamentous bacteriophage (called VP12 bearing the peptide DSQKTNPS at the N-terminus of the major coat protein pVIII) to aggregate MFT. To understand the biophysical basis of the aggregation, phage-induced aggregation of diluted MFT was measured at room temperature under varying conditions of pH, salt, detergent. Phage at concentrations of 5.0 × 10(11)/mL to 10(12)/mL induced rapid settling of the diluted MFT. The addition of sodium chloride (10 mM) lowered the concentration of phage required to induce aggregation. Since the non-ionic detergents Triton-X 100 and Tween-20, and the ionic detergent sodium deoxycholate had little effect, hydrophobic interactions do not appear to be a major contributor to the phage-induced aggregation of MFT. However, aggregation was prevented at pH values higher than 9.0 suggesting that positively charged amino acid residues are required for MFT aggregation by phage. Genetic engineering of the pVIII peptide sequence indicated that hydrogen bonding also contributes to phage-induced aggregation. In addition, replacing the basic residue lysine with an alanine in the recombinant peptide of VP12 completely prevented phage-induced aggregation. Three other phage displaying different amino acid sequences but all containing a lysine in the same position had variable aggregation efficiencies, ranging from no aggregation to rapid aggregation. We conclude that not only are the functional groups of the amino acids important, but the conformation that is adopted by the variable pVIII peptide is also important for phage-induced MFT aggregation.

Optimization of fermentation parameters in phage production using response surface methodology.

Previously, we used computer-controlled fermentation technology to improve the yield of filamentous phage produced in Escherichia coli by 10-fold (Grieco et al., Bioprocess Biosyst Eng 32:773-779, 2009). In the current study, three major fermentation parameters (temperature, dissolved oxygen [DO], and pH) were investigated using design of experiments (DOE) methodology. Response surface methodology (RSM) was employed to create a process model and determine the optimal conditions for maximal phage production. The experimental data fitted best to a quadratic model (p < 0.0001). Temperature and pH, but not DO, proved to be significant variables. The model predicted a theoretical optimal condition for maximal bacteriophage production at temperature of 28.1 °C and pH 6.9. A validation run resulted in phage production [3.49 × 10(11) transducing units (TU)/mL] comparable to the predicted value (2.86 × 10(11) TU/mL). This represented a 7-fold increase in phage production above that obtained without optimization, resulting in a 70-fold increase above that achieved by shake flask culture alone.

Effects of bacteriophage on the surface properties of chalcopyrite (CuFeS2), and phage-induced flocculation of chalcopyrite, glacial till, and oil sands tailings.

The binding of mineral-specific phage to the surface of chalcopyrite (CuFeS(2)) was investigated by using X-ray photoelectron spectroscopy and scanning Auger microscopy. These studies confirmed the elemental composition of the minerals and confirmed that bacteriophage were bound to the mineral surface. These techniques also revealed that the phage were not forming a continuous film over the entire surface of the CuFeS(2) particles, but selectively bound to the slimes coating the particles. In addition, the effect of mineral-specific phage binding to the surface of CuFeS(2) was investigated using induction time and zeta potential measurements. Bacteriophage (10(12) /mL) increased the induction time (contact time resulting in 50% particle attachment to a bubble) from ∼7.5 to ∼17 ms and reversed the zeta potential from negative to positive. In the course of performing the zeta potential measurements on particles <45 µm in diameter, phage-induced aggregation was observed. The mechanism of aggregation was explored using a range of pH (3-11) and cation concentrations. Aggregation was observed across the tested pH range and with all cations. Phage also mediated aggregation of glacial till and oil sands tailings in a dose-dependent and particle size-dependent manner. We conclude that binding of bacteriophage to the surface of CuFeS(2) does alter its surface properties.

Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic {beta}-cells.

Hephaestin (Hp) is a membrane protein with ferroxidase activity that converts Fe(II) to Fe(III) during the absorption of nutritional iron in the gut. Using anti-peptide antibodies to predicted immunogenic regions of rodent Hp, previous immunocytochemical studies in rat, mouse, and human gut tissues localized Hp to the basolateral membranes of the duodenal enterocytes where the Hp was predicted to aid in the transfer of Fe(III) to transferrin in the blood. We used a recombinant soluble form of human Hp to obtain a high-titer polyclonal antibody to Hp. This antibody was used to identify the intracellular location of Hp in human gut tissue. Our immunocytochemical studies confirmed the previous localization of Hp in human enterocytes. However, we also localized Hp to the entire length of the gastrointestinal tract, the antral portion of the stomach, and to the enteric nervous system (both the myenteric and submucous plexi). Hp was also localized to human pancreatic beta-cells. In addition to its expression in the same cells as Hp, ferroportin was also localized to the ductal cells of the exocrine pancreas. The localization of the ferroxidase Hp to the neuronal plexi and the pancreatic beta cells suggests a role for the enzymatic function of Hp in the protection of these specialized cell types from oxidative damage.

Maximizing filamentous phage yield during computer-controlled fermentation.

Filamentous phage such as M13 and fd consist of a circular, single-stranded DNA molecule surrounded by several different coat proteins. These phages have been used extensively as vectors in phage display where one of the phage coat proteins is genetically engineered to contain a unique peptide surface loop. Through these peptide sequences, a phage collection can be screened for individual phage that binds to different macromolecules or small organic and inorganic molecules. Here, we use computer-controlled bioreactors to produce large quantities of filamentous phage in the bacterial host Escherichia coli. By measuring phage yield and bacterial growth while changing the growth medium, pH and dissolved oxygen concentration, we found that the optimal conditions for phage yield were NZY medium with pH maintained at 7.4, the dO(2) held at 100% and agitation at 800 rpm. These computer-controlled fermentations result in a minimum of a tenfold higher filamentous phage production compared to standard shake flask conditions.

Biomining with bacteriophage: selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite.

During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

Helicobacter pylori infection targets adherens junction regulatory proteins and results in increased rates of migration in human gastric epithelial cells.

The human gastric pathogen Helicobacter pylori attaches to antral epithelial cells in vivo. Cultured human antral epithelial cells, AGS and NCI-N87 cell lines, were grown in the absence or presence of H. pylori and compared with respect to gene transcript levels, protein expression, organization of the actin cytoskeleton, and the regulation of cell migration. The Clontech Neurobiology array detected differentially expressed transcripts, while Western blots were used to investigate related changes in protein levels. Infection with H. pylori consistently upregulated annexin II, S100 A7, Rho-GTP, and IQGAP-1, whereas SSTR-1 was downregulated upon H. pylori infection. In the adherens junction, E-cadherin and IQGAP-1 were translocated from the plasma membrane to intracellular vesicles. The primary and NCI-N87 cells were similar with respect to cell-cell and cell-matrix adhesion and cell migratory behavior; in contrast the AGS cells were significantly different from the primary gastric epithelial cell preparations, and thus caution must be used when using this cell line for studies of gastric disease. These studies demonstrate a correlation between H. pylori infection and alterations to epithelial cell adhesion molecules, including increased levels of Rho-GTP and cell migration. These data indicate that destabilizing epithelial cell adherence is one of the factors increasing the risk of H. pylori-infected individuals developing gastric cancer.