Romany Abskharon

Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP(C), into its misfolded oligomeric form, known as prion or PrP(Sc). The canonical human PrP(C) (HuPrP) fold features an unstructured N-terminal part (residues 23-124) and a well-defined C-terminal globular domain (residues 125-231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113-120) plays an important role in the conversion to PrP(Sc). The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel β-sheet with the β1 and β2 strands, demonstrating that the full-length HuPrP(C) can adopt a more elaborate β0-β1-α1-β2-α2-α3 structural organization than the canonical β1-α1-β2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP(C) into PrP(Sc).

Isolation, characterization of heavy metal resistant strain of Pseudomonas aeruginosa isolated from polluted sites in Assiut city, Egypt

Sixty six isolates of Pseudomonas spp. were isolated from wastewater of El‐Malah canal located in Assiut, Egypt and were checked for their heavy metal tolerance. One isolate has tested for its multiple metal resistances and found to be plasmid mediated with molecular weight 27 Kb for nickel and lead. It was identified as Pseudomonas aeruginosa ASU 6a. Its minimal inhibitory concentration (MIC) for Cu2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+and Pb2+ were 6.3, 5.9, 6.8, 9.2, 5.8, 4.4, and 3.1 mM, respectively. Growth kinetics and the maximum adsorption capacities were determined under Ni2+ and Pb2+ stress. The latter heavy metals induced potassium efflux and were used as indicator for plasma membrane permeabilization. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A novel expression system for production of soluble prion proteins in E. coli

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

Toxicity assessment using different bioassays and microbial biosensors

Toxicity assessment of water streams, wastewater, and contaminated sediments, is a very important part of environmental pollution monitoring. Evaluation of biological effects using a rapid, sensitive and cost effective method can indicate specific information on ecotoxicity assessment. Recently, different biological assays for toxicity assessment based on higher and lower organisms such as fish, invertebrates, plants and algal cells, and microbial bioassays have been used. This review focuses on microbial biosensors as an analytical device for environmental, food, and biomedical applications. Different techniques which are commonly used in microbial biosensing include amperometry, potentiometry, conductometry, voltammetry, microbial fuel cells, fluorescence, bioluminescence, and colorimetry. Examples of the use of different microbial biosensors in assessing a variety of environments are summarized.

Recombinant human prion protein inhibits prion propagation in vitro.

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrPSc, but not PrPC, suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrPC with PrPSc. Our findings suggest a new avenue for treating prion diseases, in which a patients own unglycosylated and anchorless PrP is used to inhibit PrPSc propagation without inducing immune response side effects.

The role of the unusual threonine string in the conversion of prion protein

The conversion of normal prion protein (PrP) into pathogenic PrP conformers is central to prion disease, but the mechanism remains unclear. The α-helix 2 of PrP contains a string of four threonines, which is unusual due to the high propensity of threonine to form β-sheets. This structural feature was proposed as the basis for initiating PrP conversion, but experimental results have been conflicting. We studied the role of the threonine string on PrP conversion by analyzing mouse Prnpa and Prnpb polymorphism that contains a polymorphic residue at the beginning of the threonine string, and PrP mutants in which threonine 191 was replaced by valine, alanine, or proline. The PMCA (protein misfolding cyclic amplification) assay was able to recapitulate the in vivo transmission barrier between PrPa and PrPb. Relative to PMCA, the amyloid fibril growth assay is less restrictive, but it did reflect certain properties of in vivo prion transmission. Our results suggest a plausible theory explaining the apparently contradictory results in the role of the threonine string in PrP conversion and provide novel insights into the complicated relationship among PrP stability, seeded conformational change, and prion structure, which is critical for understanding the molecular basis of prion infectivity.

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein.

Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=30.04, b=37.15, c=83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors

BackgroundThe infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease.ResultsTo explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion.ConclusionsOur results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.