DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Systematic review and meta-analysis of randomised trials and cohort studies of mycophenolate mofetil in lupus nephritis

Mycophenolate mofetil (MMF) is an immunosuppressant drug being used for induction and maintenance of remission of lupus nephritis in systemic lupus erythematosus. Evidence about its use was sought from full publications and abstracts of randomised trials and cohort studies by using a variety of search strategies. Efficacy and adverse event outcomes were sought. Five randomised trials enrolled patients with World Health Organization (WHO) class III, IV, or V (mostly IV) lupus nephritis, predominantly comparing MMF (1 to 3 g daily) with cyclophosphamide and steroid. Complete response and complete or partial response was significantly more frequent with MMF than with cyclophosphamide, with numbers needed to treat of 8 (95% confidence interval 4.3 to 60) to induce one additional complete or partial response, with wide confidence intervals. Death was reported less frequently with MMF (0.7%, 1 death in 152 patients) than with cyclophosphamide (7.8%, 12 deaths in 154 patients), with a number needed to treat to prevent (NNTp) one death of 14 (8 to 48). Hospital admission was also lower with MMF (1.7% versus 15%; NNTp 7.4 [4.8 to 16]). Serious infections, leucopaenia, amenorrhoea, and hair loss were all significantly less frequent with MMF than with cyclophosphamide, but diarrhoea was significantly more common with MMF. Ten of 18 cohort studies enrolled only patients with lupus nephritis (author-defined or WHO class III to V). Seven of these 10 reported that complete or partial response with MMF (mostly 1 or 2 g daily) with steroid occurred in 121/151 (80%) and that treatment failure or no response occurred in 30/151 (20%). Adverse events were generally similar in cohort studies with and without only patients with lupus nephritis. In all 18 cohorts, gastrointestinal adverse events (diarrhoea, nausea, vomiting) occurred in 30%, infection in 23%, and serious infection in 4.3%. Adverse event discontinuations occurred in 14% and lack of efficacy occurred in 10%. There was a single death with MMF, a mortality rate over the course of 1 year of approximately 0.2%. The results form a basis on which to plan future studies and provide a guide for the use of MMF in lupus nephritis until results of larger studies are available. At least one such study is under way.     

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β(2) microglobulin (β(2)m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β(2)m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation and diverting the course of assembly to the formation of spherical aggregates. The results demonstrate the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers toward alternative assembly pathways.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.     

Dynamics in the unfolded state of beta2-microglobulin studied by NMR

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.     

Spatial distribution of lead in enamel and coronal dentine of wistar rats

Lead is one of the most hazardous environmental toxins known. The assessment of lead in dental hard tissues is important in the understanding of its toxic effects on oral tissues and in estimating exposure and body burden in individuals exposed to lead from the environment. However, current information on the uptake and distribution of lead in enamel and dentine is limited. The aim of this project was to study, at high resolution, the spatial distribution of lead in enamel and coronal dentine using an experimental rat model. A dose of 40 mg/L of lead nitrate was administered to pregnant female rats during the periods of gestation and lactation through drinking water. First mandibular molar teeth were removed from their 15-d-old pups and the distribution of lead was studied using a nuclear microprobe (NMP). The distribution of lead in enamel and coronal dentine showed four distinct zones with significantly different mean lead concentrations (p<0.05). High levels of lead were observed in the superficial regions of enamel and in the dentine directly adjacent to the pulp. Additionally, the results confirmed that the NMP is capable of mapping the distribution of lead in teeth at micron resolutions with a detection limit of approx 1 microg/g.