N-terminal helix-cap in α-helix 2 modulates ß-state misfolding in rabbit and hamster prion proteins.

Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the ß-state. A helix-cap motif in the ß2-α2-loop of native α-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on ß-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45-1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrP(C). They also alter the ß-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into ß-state buffer conditions causes quick conversion to ß-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the ß2-α2 helix-cap affects refolding to the ß-state and subsequently, may influence susceptibility to prion disease.

Adaptor protein self-assembly drives the control of a cullin-RING ubiquitin ligase.

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

Studies on the biology, chemotherapy and distribution of warble fly in Pakistan.

This paper presents data on the prevalence, biology and control of warble fly infestation (WFI) in cattle and goats in Pakistan. A questionnaire for obtaining information on biology and prevalence was circulated amongst field veterinary staff and livestock farmers in all five provinces (Punjab, Sindh, Balochistan, Khyber Pakhtunkhwa [KPK] and Gilgit-Baltistan) and in the Federally Administered Tribal Areas of the country. A total of 1,019 questionnaires were received (Punjab = 296, Sindh = 246, KPK = 318, Balochistan = 151, Gilgit-Baltistan = 8). Warble fly infestation was reported from each province and from the federally administered tribal areas, particularly from hilly, semi-hilly and sandy desert areas (the Cholistan desert, which adjoins the Rehim Yar Khan, Bahawalpur and Bahawalnagar districts, and the Nara area of Sanghar district). Warbles (nodules) started appearing on the backs of the infested animals from September through December and disappeared from October through March. The prevalence of WFI varied from 5% to 75%. It was highest in hilly areas and gradually decreased towards the plains. A map was developed of warble fly-infested areas. Four field trials were conducted to study the efficacy of different drugs indicated for the control of warble fly infestation. A total of 2,094 cattle and 3,876 goats were given five different injectables (avermectins); namely, Ivomec, Endectin, Euvectin, Dectomax and Promectin (ivermectin) during the first three weeks of September. A control group was given normal saline. All the medicines were found to be effective in controlling infestation.

Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological ß-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic ß-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether ß-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a ß-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the ß-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Structural factors underlying the species barrier and susceptibility to infection in prion disease.

The term prion disease describes a group of fatal neurodegenerative diseases that are believed to be caused by the pathogenic misfolding of a host cell protein, PrP. Susceptibility to prion disease differs between species and incubation periods before symptom onset can change dramatically when infectious prion strains are transmitted between species. This effect is referred to as the species or transmission barrier. Prion strains represent different structures of PrPSc and the conformational selection model proposes that the source of theses barriers is the preferential incorporation of PrP from a given species into only a subset of PrPSc structures of another species. The basis of this preferential incorporation is predicted to reside in subtle structural differences in PrP from varying species. The overall fold of PrP is highly conserved among species, but small differences in the amino acid sequence give rise to structural variability. In particular, the loop between the second beta-strand and the second alpha-helix has shown structural variability between species, with loop mobility correlating with resistance to prion disease. Single amino acid polymorphisms in PrP within a species can also affect prion susceptibility, but do not appear to drastically alter the biophysical properties of the native form. These polymorphisms affect the propensity of self-association, in recombinant PrP, to form beta-sheet enriched, oligomeric, and amyloid-like forms. These results indicate that the major factor in determining susceptibility to prion disease is the ability of PrP to adopt these misfolded forms by promoting conformational change and self association.

The epidemiology of peste des petits ruminants in Pakistan.

Peste des petits ruminants (PPR) is an acute and highly contagious viral disease of small ruminants, which is newly emerging in Pakistan. Information provided by participatory disease surveillance teams was used to develop a database for PPR outbreaks in Pakistan. Twenty-four villages were selected throughout the country and field investigations were conducted in each village to study the dynamics of the disease. In each area, flocks with a history of PPR were identified and serological sampling was conducted. Some 1,463 small ruminants (sheep and goats) were sampled and 1,096 tested positive for the presence of antibodies against PPR. These results clearly indicate that PPR is prevalent throughout Pakistan.

Integrated approach for improving small scale market oriented dairy systems in Pakistan: participatory rural appraisal and economic opportunity survey.

Livestock production is an integral part of the rain-fed and irrigated agriculture system in Pakistan. Animal production is closely interlinked with the cropping systems and play a crucial role in the rural economy. Participatory rural appraisals and economic opportunity surveys were conducted in two ecological zones (irrigated and rain-fed) and two dairy production systems (peri-urban and mixed livestock). Major constraints in animal health, nutrition and reproduction were identified and interventions were suggested to overcome these difficulties. The economic opportunity survey revealed that maximum opportunity to enhance farmers' income is to increase milk production per day per animal, which can be accomplished through coordinated improvements in nutrition, reproduction and genetics.