Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.     

Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.     

Common Cuckoo Cuculus canorus and host behaviour at Reed Warbler Acrocephalus scirpaceus nests

This study is based on continuous video recordings made at 53 Reed Warbler Acrocephalus scirpaceus nests each day during the laying period. Egg-laying by the Common Cuckoo Cuculus canorus was recorded in 14 (26.4%) of these nests. By analysing the activity of the host birds around and at the nest, we found that this is probably not the only cue used by the Common Cuckoo when locating suitable nests to parasitize. Furthermore, in most cases there was no significant difference between the length of time the host birds spent at the nest in the morning and afternoon, thus providing little support for the hypothesis that the Common Cuckoo lays in the afternoon because it is less likely to be seen by the nest owners then. Parasitized Reed Warblers rejected the Common Cuckoo egg more frequently when they observed the parasite at their nests. However, contrary to what should be expected, most Common Cuckoos laid their eggs in the presence of the host(s), and in general their egg-laying behaviour (for example duration of stay at the nest) was less secretive than described earlier. When partially depredating host clutches, Cuckoos showed the same behavioural pattern at parasitized and unparasitized nests, indicating that the latter may act as a potential reserve for egg-laying.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.     

Demographic costs of inbreeding revealed by sex-specific genetic rescue effects

Background  

Inbreeding can slow population growth and elevate extinction risk. A small number of unrelated immigrants to an inbred population can substantially reduce inbreeding and improve fitness, but little attention has been paid to the sex-specific effects of immigrants on such "genetic rescue". We conducted two subsequent experiments to investigate demographic consequences of inbreeding and genetic rescue in guppies.     

Expression of growth hormone-releasing hormone (GHRH) and splice variant of GHRH receptors in normal mouse tissues

Growth hormone-releasing hormone (GHRH) stimulates the production and release of growth hormone in the pituitary and induces cell proliferation in a variety of peripheral tissues and tumors. These extrapituitary effects of GHRH are in many cases mediated by a splice variant of GHRH receptor designated SV1 that differs from the pituitary GHRH receptor in a small portion of its amino-terminal region. While SV1 has been detected in several primary tumors and many cancer cell lines its expression in normal tissues remains unclear. In this study we report the results of an immunohistochemical analysis for SV1 and GHRH expression in normal mouse tissues. For the detection of SV1 immunoreactivity we used a polyclonal antiserum against segments 1-25 of the SV1 receptor protein. Mouse heart, colon, lungs, small intestine, stomach and kidneys exhibited increased SV1 immunoreactivity. These tissues were also positive for GHRH expression, however, tissues such as the endometrium were positive only for GHRH and not for SV1 expression. On the contrary, testis were positive for SV1 and not for GHRH expression. These results indicate that SV1 may play a role in normal physiology.     

Identification of Cultured Brachionus Rotifers Based on RFLP and SSCP Screening

The marine finfish industry worldwide depends greatly on the mass culture of Brachionus rotifers. Recently, molecular data have revealed a more complicated view about the species status of Brachionus rotifers than previous mainly morphological assessments. Under this view, Brachionus rotifers are comprised of many morphologically similar, albeit genetically differentiated, cryptic members of larger groups. A redefinition of the cultured rotifer species/biotypes is therefore needed if aquaculture is to reach higher levels of standardization and predictability. In this work, restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) methods are applied to the COI and 16S rRNA mitochondrial genes. A detailed COI restriction map was constructed, using sequence data from all known representatives of Brachionus phylogroups. Therefore, it is the first time that such an extended restriction database has been produced. Several restriction endonucleases are proposed for the discrimination of the different Brachionus species/biotypes. Furthermore, eight different SSCP gel alleles are described for the 16S region. Using these data, five Brachionus species/biotypes were identified in 78 samples collected from laboratories and hatcheries around the world. Spiros Papakostas and Stefania Dooms contributed equally to this work.