Functional analysis of the sheep Wilson disease protein (sATP7B) in CHO cells

In this study we investigated the function of the sheep orthologue of ATP7B (sATP7B), the protein affected in the human copper toxicosis disorder Wilson disease. Two forms of sATP7B are found in the sheep, a ‘normal’ form and one with an alternate N terminus, both of which were expressed in CHO-K1 cells. Cells expressing either form of sATP7B were more resistant to copper than the parental CHO-K1 cells. Subcellular localisation studies showed that both forms of sATP7B were similarly located in the trans-Golgi network (TGN). When the extracellular copper concentration was increased, each form of sATP7B redistributed to a punctate, vesicular compartment that extended throughout the cytoplasm. Both forms of sATP7B recycled to the perinuclear location within one hour when the cells were subsequently incubated in basal medium. After treatment of cells with bafilomycin A1 sATP7B accumulated in cytoplasmic vesicles, implying that ATP7B continuously recycles via the endocytic pathway. These results suggest that both forms of sATP7B are functional copper-transport proteins and that the intracellular location and trafficking of the sheep protein within the cell also appears normal.

2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human A1 adenosine receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5- dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro 4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino- 3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA¹AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist- A₁AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH₂)_n- bridge, n ) 3 < n ) 4 < n ) 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA₁AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Structure and phase stability of NiMnGa ferromagnetic shape memory alloys by experimental and ab-initio techniques

This paper summarizes some of our recent results on crystal structure, microstructure, orientation relationship between martensitic variants and crystallographic features of martensitic transformation in Ni-Mn-Ga FSMAs. It was shown that Ni53Mn25Ga22 has a tetragonal I4/mmm martensitic structure at room temperature. The neighboring martensitic variants in Ni53Mn25Ga22 have a compound twinning relationship with the twinning elements K1={112}, K2={11-2}, η1=<11-1>, η2=<111>, P={1-10} and s=0.379. The ratio of the relative amounts of twins within the same initial austenite grain is ~1.70. The main orientation relationship between austenite and martensite is Kurdjumov-Sachs (K-S) relationship. Based on the crystallographic phenomenological theory, the calculated habit plane is {0.690 -0.102 0.716}A (5.95° from {101}A), and the magnitude, direction and shear angle of the macroscopic transformation shear are 0.121, <-0.709 0.105 0.698>A (6.04° from <-101>A) and 6.88°, respectively.

Influence of orientation on twin nucleation and growth at low strains in a magnesium alloy

The resolved shear stress is believed to play an important role in twin formation. The present study tests this idea for an extruded magnesium alloy by examining "tension" twinning in different grain orientations. Electron backscatter diffraction analysis is employed for alloy AZ31 tested in compression along the extrusion axis to strains between 0.008 and 0.015. For heavily twinned grains, it is seen that twinning occurs on 2.3 twin systems per grain on average. The active systems are also most commonly those with, or very near to, the highest Schmid factor. The most active system in multiply twinned grains accounts on average for ∼0.6 of the twinning events. In addition, it is found that the twin habit plane falls within 6° of the K1 plane. Orientations with the highest Schmid factors (0.45-0.5) for twinning display twin aspect ratios greater by ∼40% and twin number densities greater by ∼10 times than orientations with maximum Schmid factors for twinning of 0.15-0.2. Thus the Schmid factor for twinning is seen to affect nucleation more than thickening in the present material. Viscoplastic crystal plasticity simulations are employed to obtain approximations for the resolved shear stress. Both the twin aspect ratio and number density correlate quite well with this term. The effect of the former can be assumed to be linear and that of the latter follows a power law with exponent ∼13. Increased aspect ratios and number densities are seen at low Schmid factors and this may relate to stress fluctuations, caused most probably in the present material by the stress fields at the tips of blocked twins. Overall, it is evident that the dominance of twinning on high Schmid factor systems is preserved at the low strains examined in the present work, despite the stress fluctuations known to be present. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Complete structure of an epithelial keratin dimer: implications for intermediate filament assembly

Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general.

Cross-strand disulfides in the non-hydrogen bonding site of antiparallel β-sheet (aCSDns): poised for biological switching

Forbidden disulfides are stressed disulfides found in recognisable protein contexts previously defined as structurally forbidden. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. The meta-stability of forbidden disulfides makes them likely candidates as redox switches. Here we mined the Protein Data Bank for examples of the most common forbidden disulfide, the aCSDn. This is a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen bonded moieties are directed away from each other. We grouped these aCSDns into homologous clusters and performed an extensive physicochemical and informatic analysis of the examples found. We estimated their torsional energies using quantum chemical calculations and studied differences between the preferred conformations of the computational model and disulfides found in solved protein structures to understand the interaction between the forces imposed by the disulfide linkage and typical constraints of the surrounding β-sheet. In particular, we assessed the twisting, shearing and buckling of aCSDn-containing β-sheets, as well as the structural and energetic relaxation when hydrogen bonds in the motif are broken. We show the strong preference of aCSDns for the right-handed staple conformation likely arises from its compatibility with the twist, shear and Cα separation of canonical β-sheet. The disulfide can be accommodated with minimal distortion of the sheet, with almost all the strain present as torsional strain within the disulfide itself. For each aCSDn cluster, we summarise the structural and strain data, taxonomic conservation and any evidence of redox activity. aCSDns are known substrates of thioredoxin-like enzymes. This, together with their meta-stability, means they are ideally suited to biological switching roles and are likely to play important roles in the molecular pathways of oxidative stress.