Quantification of Conformational Heterogeneity and its Role in Protein Aggregation and Unfolding

Proteins can exhibit significant conformational heterogeneity either under denaturing conditions or in aqueous solutions. The latter is true for a class of proteins whose sequences predispose them to form heterogeneous ensembles of conformations. Characterization of conformational heterogeneity in a protein ensemble requires the quantification of the amplitudes of spontaneous fluctuations in conjunction with information regarding coarse grain measures that report on the average sizes, shapes, and densities. This often demands multiplexed experimental approaches whose readouts are interpreted or annotated using ensembles drawn from atomistic or coarse grain computational simulations. Efforts to characterize conformational heterogeneity contribute directly to our understanding of disorder-to-order transitions in protein folding and self-assembly. These efforts are also crucial to our understanding of the heterotypic interactions involving intrinsically disordered proteins and non-native states of well-folded proteins. These heterotypic interactions are important in signal transduction and the regulation of protein homeostasis. The onset and progression of several systemic and neurodegenerative conformational diseases are linked to the nature and degree of conformational heterogeneity in specific proteins or proteolytic products of proteins. This thesis work focuses on the quantitative characterization of conformational heterogeneity in simulated ensembles of inducibly unfolded and intrinsically disordered proteins. Advances in nuclear magnetic resonance spectroscopy afford the possibility of detailed measurements of inter-residue distances and modulations to the relaxation dynamics of paramagnetic spins that are inserted as probes into a protein. These state-of-the-art measurements show interesting features within denatured state ensembles that cannot be explained using canonical random coil models. Here, we use computer simulations to generate plausible facsimiles of denatured state ensembles that reproduce experimental data and demonstrate that the ensembles that are consistent with the data are characterized by the presence of low-likelihood, long-range intra-chain contacts between hydrophobic groups. When placed in the context of sequence conservation information, it appears that these contacts act as gatekeepers that protect proteins from the deleterious consequences of protein aggregation by sequestering hydrophobic groups in an assortment of intra-chain long-range contacts. We also characterize the nature and degree of conformational heterogeneity in glutamine- and asparagine-rich containing systems. These efforts lead to insights regarding the role of conformational heterogeneity in mediating intermolecular associations that are implicated in aggregation and self-assembly of these systems. Analysis of results from atomistic simulations leads to a phenomenological model for the modulation of conformational heterogeneity and degeneracies of intermolecular interactions by naturally occurring sequences that flank polyglutamine domains. Finally, we develop a formal order parameter to quantify the conformational heterogeneity in simulated ensembles of proteins. When combined with measures of density and fluctuations thereof, it can be used to provide a complete description of the degree and nature of conformational heterogeneity in different ensembles, thus affording the ability to compare different ensembles to each other while also providing a way to categorize conformational transitions

Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens

What makes you not a Sikh? : a preliminary mapping of values

This study sets out to establish which Sikh values contrasted with or were shared by non-Sikh adolescents. A survey of attitude toward a variety of Sikh values was fielded in a sample of 364 non-Sikh schoolchildren aged between 13 and 15 in London. Values where attitudes were least positive concerned Sikh duties/code of conduct, festivals, rituals, prayer Gurdwara attendance, listening to scripture recitation, the amrit initiation. Sikh values empathized with by non-Sikhs concerned family pride, charity, easy access to ordination and Gurdwaras, maintaining the five Ks, seeing God in all things, abstaining from meat and alcohol and belief in the stories of Guru Nanak. Further significant differences of attitude toward Sikhism were found in comparisons by sex, age and religious affiliation. Findings are applied to teaching Sikhism to pupils of no faith adherence. The study recommends the extension of values mapping to specifically Sikh populations

Efficacy of three neuroprotective drugs in secondary progressive multiple sclerosis (MS-SMART):a phase 2b, multiarm, double-blind, randomised placebo-controlled trial

Neurodegeneration is the pathological substrate that causes major disability in secondary progressive multiple sclerosis. A synthesis of preclinical and clinical research identified three neuroprotective drugs acting on different axonal pathobiologies. We aimed to test the efficacy of these drugs in an efficient manner with respect to time, cost, and patient resource. Methods: We did a phase 2b, multiarm, parallel group, double-blind, randomised placebo-controlled trial at 13 clinical neuroscience centres in the UK. We recruited patients (aged 25-65 years) with secondary progressive multiple sclerosis who were not on disease-modifying treatment and who had an Expanded Disability Status Scale (EDSS) score of 4·0-6·5. Participants were randomly assigned (1:1:1:1) at baseline, by a research nurse using a centralised web-based service, to receive twice-daily oral treatment of either amiloride 5 mg, fluoxetine 20 mg, riluzole 50 mg, or placebo for 96 weeks. The randomisation procedure included minimisation based on sex, age, EDSS score at randomisation, and trial site. Capsules were identical in appearance to achieve masking. Patients, investigators, and MRI readers were unaware of treatment allocation. The primary outcome measure was volumetric MRI percentage brain volume change (PBVC) from baseline to 96 weeks, analysed using multiple regression, adjusting for baseline normalised brain volume and minimisation criteria. The primary analysis was a complete-case analysis based on the intention-to-treat population (all patients with data at week 96). This trial is registered with ClinicalTrials.gov, NCT01910259

Bone histomorphometric measures of physical activity in children from Medieval England

Objectives: Histomorphometric studies show consistent links between physical activity patterns and the microstructure underlying the size and shape of bone. Here we adopt a combined bone approach to explore variation in microstructure of ribs and humeri related to physical activity and historical records of manual labor in skeletal samples of children (n=175) from medieval England. The humerus reflects greater biomechanically induced microstructural variation than the rib which is used here as a control. Variation in microstructure is sought between regions in England (Canterbury, York, Newcastle), and between high- and low-status children from Canterbury. Materials and Methods: Thin-sections were prepared from the humerus or rib and features of bone remodeling were recorded using high-resolution microscopy and image analysis software. Results: The density and size of secondary osteons in the humerus differed significantly in children from Canterbury when compared to those from York and Newcastle. Amongst the older children, secondary osteon circularity and diameter differed significantly between higher and lower status children. Discussion: By applying bone remodeling principles to the histomorphometric data we infer that medieval children in Canterbury engaged in less physically demanding activities than children from York or Newcastle. Within Canterbury, high-status and low-status children experienced similar biomechanical loading until around seven years of age. After this age low-status children performed activities that resulted in more habitual loading on their arm bones than the high-status children. This inferred change in physical activity is consistent with historical textual evidence that describes children entering the work force at this age

Iron-overload-related disease in HFE hereditary hemochromatosis

Background: Most persons who are homozygous for C282Y, the HFE allele most commonly asssociated with hereditary hemochromatosis, have elevated levels of serum ferritin and transferrin saturation. Diseases related to iron overload develop in some C282Y homozygotes, but the extent of the risk is controversial. Methods: We assessed HFE mutations in 31,192 persons of northern European descent between the ages of 40 and 69 years who participated in the Melbourne Collaborative Cohort Study and were followed for an average of 12 years. In a random sample of 1438 subjects stratified according to HFE genotype, including all 203 C282Y homozygotes (of whom 108 were women and 95 were men), we obtained clinical and biochemical data, including two sets of iron measurements performed 12 years apart. Disease related to iron overload was defined as documented iron overload and one or more of the following conditions: cirrhosis, liver fibrosis, hepatocellular carcinoma, elevated aminotransferase levels, physician-diagnosed symptomatic hemochromatosis, and arthropathy of the second and third metacarpophalangeal joints. Results: The proportion of C282Y homozygotes with documented iron-overload-related disease was 28.4% (95% confidence interval [CI], 18.8 to 40.2) for men and 1.2% (95% CI, 0.03 to 6.5) for women. Only one non-C282Y homozygote (a compound heterozygote) had documented iron-overload-related disease. Male C282Y homozygotes with a serum ferritin level of 1000 μg per liter or more were more likely to report fatigue, use of arthritis medicine, and a history of liver disease than were men who had the wild-type gene. Conclusions: In persons who are homozygous for the C282Y mutation, iron-overload-related disease developed in a substantial proportion of men but in a small proportion of women

Genome-Wide Association and Trans-ethnic Meta-Analysis for Advanced Diabetic Kidney Disease: Family Investigation of Nephropathy and Diabetes (FIND)

Diabetic kidney disease (DKD) is the most common etiology of chronic kidney disease (CKD) in the industrialized world and accounts for much of the excess mortality in patients with diabetes mellitus. Approximately 45% of U.S. patients with incident end-stage kidney disease (ESKD) have DKD. Independent of glycemic control, DKD aggregates in families and has higher incidence rates in African, Mexican, and American Indian ancestral groups relative to European populations. The Family Investigation of Nephropathy and Diabetes (FIND) performed a genome-wide association study (GWAS) contrasting 6,197 unrelated individuals with advanced DKD with healthy and diabetic individuals lacking nephropathy of European American, African American, Mexican American, or American Indian ancestry. A large-scale replication and trans-ethnic meta-analysis included 7,539 additional European American, African American and American Indian DKD cases and non-nephropathy controls. Within ethnic group meta-analysis of discovery GWAS and replication set results identified genome-wide significant evidence for association between DKD and rs12523822 on chromosome 6q25.2 in American Indians (P = 5.74x10-9). The strongest signal of association in the trans-ethnic meta-analysis was with a SNP in strong linkage disequilibrium with rs12523822 (rs955333; P = 1.31x10-8), with directionally consistent results across ethnic groups. These 6q25.2 SNPs are located between the SCAF8 and CNKSR3 genes, a region with DKD relevant changes in gene expression and an eQTL with IPCEF1, a gene co-translated with CNKSR3. Several other SNPs demonstrated suggestive evidence of association with DKD, within and across populations. These data identify a novel DKD susceptibility locus with consistent directions of effect across diverse ancestral groups and provide insight into the genetic architecture of DKD

Dendritic Spikes Amplify the Synaptic Signal to Enhance Detection of Motion in a Simulation of the Direction-Selective Ganglion Cell

The On-Off direction-selective ganglion cell (DSGC) in mammalian retinas responds most strongly to a stimulus moving in a specific direction. The DSGC initiates spikes in its dendritic tree, which are thought to propagate to the soma with high probability. Both dendritic and somatic spikes in the DSGC display strong directional tuning, whereas somatic PSPs (postsynaptic potentials) are only weakly directional, indicating that spike generation includes marked enhancement of the directional signal. We used a realistic computational model based on anatomical and physiological measurements to determine the source of the enhancement. Our results indicate that the DSGC dendritic tree is partitioned into separate electrotonic regions, each summing its local excitatory and inhibitory synaptic inputs to initiate spikes. Within each local region the local spike threshold nonlinearly amplifies the preferred response over the null response on the basis of PSP amplitude. Using inhibitory conductances previously measured in DSGCs, the simulation results showed that inhibition is only sufficient to prevent spike initiation and cannot affect spike propagation. Therefore, inhibition will only act locally within the dendritic arbor. We identified the role of three mechanisms that generate directional selectivity (DS) in the local dendritic regions. First, a mechanism for DS intrinsic to the dendritic structure of the DSGC enhances DS on the null side of the cell's dendritic tree and weakens it on the preferred side. Second, spatially offset postsynaptic inhibition generates robust DS in the isolated dendritic tips but weak DS near the soma. Third, presynaptic DS is apparently necessary because it is more robust across the dendritic tree. The pre- and postsynaptic mechanisms together can overcome the local intrinsic DS. These local dendritic mechanisms can perform independent nonlinear computations to make a decision, and there could be analogous mechanisms within cortical circuitry

Genome-wide association studies identify CHRNA5/3 and HTR4 in the development of airflow obstruction

RATIONALE: Genome-wide association studies (GWAS) have identified loci influencing lung function, but fewer genes influencing chronic obstructive pulmonary disease (COPD) are known.OBJECTIVES: Perform meta-analyses of GWAS for airflow obstruction, a key pathophysiologic characteristic of COPD assessed by spirometry, in population-based cohorts examining all participants, ever smokers, never smokers, asthma-free participants, and more severe cases.METHODS: Fifteen cohorts were studied for discovery (3,368 affected; 29,507 unaffected), and a population-based family study and a meta-analysis of case-control studies were used for replication and regional follow-up (3,837 cases; 4,479 control subjects). Airflow obstruction was defined as FEV(1) and its ratio to FVC (FEV(1)/FVC) both less than their respective lower limits of normal as determined by published reference equations.MEASUREMENTS AND MAIN RESULTS: The discovery meta-analyses identified one region on chromosome 15q25.1 meeting genome-wide significance in ever smokers that includes AGPHD1, IREB2, and CHRNA5/CHRNA3 genes. The region was also modestly associated among never smokers. Gene expression studies confirmed the presence of CHRNA5/3 in lung, airway smooth muscle, and bronchial epithelial cells. A single-nucleotide polymorphism in HTR4, a gene previously related to FEV(1)/FVC, achieved genome-wide statistical significance in combined meta-analysis. Top single-nucleotide polymorphisms in ADAM19, RARB, PPAP2B, and ADAMTS19 were nominally replicated in the COPD meta-analysis.CONCLUSIONS: These results suggest an important role for the CHRNA5/3 region as a genetic risk factor for airflow obstruction that may be independent of smoking and implicate the HTR4 gene in the etiology of airflow obstruction.</p

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe