Fibril formation of the rabbit/human/bovine prion proteins

Prion diseases are infectious fatal neurodegenerative diseases including Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. The misfolding and conversion of cellular PrP in such mammals into pathogenic PrP is believed to be the key procedure. Rabbits are among the few mammalian species that exhibit resistance to prion diseases, but little is known about the molecular mechanism underlying such resistance. Here, we report that the crowding agents Ficoll 70 and dextran 70 have different effects on fibrillization of the recombinant full-length PrPs from different species: although these agents dramatically promote fibril formation of the proteins from human and cow, they significantly inhibit fibrillization of the rabbit protein by stabilizing its native state. We also find that fibrils formed by the rabbit protein contain less β-sheet structure and more α-helix structure than those formed by the proteins from human and cow. In addition, amyloid fibrils formed by the rabbit protein do not generate a proteinase K-resistant fragment of 15–16-kDa, but those formed by the proteins from human and cow generate such proteinase K-resistant fragments. Together, these results suggest that the strong inhibition of fibrillization of the rabbit PrP by the crowded physiological environment and the absence of such a protease-resistant fragment for the rabbit protein could be two of the reasons why rabbits are resistant to prion diseases.     

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.     

Close is not enough: SNARE-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors

Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15–55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415–421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.     

Peptide-binding motif of HLA-A*6603

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.     

An α-Amylase Homologue,aah3, Encodes a GPI-Anchored Membrane Protein Required for Cell Wall Integrity and Morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four α-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3 ⁺ resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3 ⁺ deletion, but the ascal wall of aah3Δ cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Δ cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

Influence of the lubricant and the alloy on the wear behaviour of attachments

Gerodontology 2010; doi: 10.1111/j.1741‐2358.2009.00352.x
Influence of the lubricant and the alloy on the wear behaviour of attachments Objectives: Wear of attachments leads to a loss of retention and reduces the function of overdentures. This study evaluated the retention force changes of an attachment system for overdentures. The influence of the lubricant and the alloy on wear constancy was examined. Methods: Cylindrical anchors of the Dalbo^®‐Z system were tested (Cendres+Métaux SA). Three groups of alloy‐lubricant combinations were generated 1.Elitor^®/NaCl‐solution (EN) 2.Elitor^®/Glandosane^®aquadest. (EG) and 3.Valor^®/Glandosane^®/aquadest. (VG). Ten samples of each group were subjected to 10 000 insertion–separation cycles. Results: For the EN‐group, this led to a large increase in retention force. The EG‐ and VG‐group showed a constant decrease after an initial increase in retention force at the beginning of the wear simulation. The change of the alloy caused no statistically significant differences. The use of a more viscous lubricant reduced the retention force increase significantly. Conclusions: The use of a lubricant which simulates clinical conditions is an absolute need for wear simulation because the retention force changes are influenced enormously. The change of the alloy at the Dalbo^®‐Z system did not influence the wear behaviour. As a slight decrease in retention force was recorded, it is useful for an attachment system to allow compensation with an adjustable matrix.     

Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission.     

Crystal structure of the ligand binding domain of netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca²⁺ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.     

Technical aspects of functional proteomics in plants

Since the completion of genome sequences of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. This analysis is achieved by separation and identification of proteins, determination of their function and functional network, and construction of an appropriate database. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis, nano-liquid chromatography and mass spectrometry, have rapidly been achieved. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. These techniques have provided the possibility of high-throughput analysis of function and functional network of proteins in plants. However, to cope with the huge information emerging from proteome analyses, more sophisticated techniques and software are essential. The development and adaptation of such techniques will ease analyses of protein profiling, identification of post-translational modifications and protein-protein interaction, which are vital for elucidation of the protein functions.