SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

Centromeric histone H3, CENP-A^Cse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A^Cse4 restricts its localization to centromeres. Mislocalization of CENP-A^Cse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.     

Does perspective matter? A case study comparing Eulerian and Lagrangian estimates of common murre (Uria aalge) distributions

Studies estimating species' distributions require information about animal locations in space and time. Location data can be collected using surveys within a predetermined frame of reference (i.e., Eulerian sampling) or from animal‐borne tracking devices (i.e., Lagrangian sampling). Integration of observations obtained from Eulerian and Lagrangian perspectives can provide insights into animal movement and habitat use. However, contemporaneous data from both perspectives are rarely available, making examination of biases associated with each sampling approach difficult. We compared distributions of a mobile seabird observed concurrently from ship, aerial, and satellite tag surveys during May, June, and July 2012 in the northern California Current. We calculated utilization distributions to quantify and compare variability in common murre (Uria aalge) space use and examine how sampling perspective and platform influence observed patterns. Spatial distributions of murres were similar in May, regardless of sampling perspective. Greatest densities occurred in coastal waters off southern Washington and northern Oregon, near large murre colonies and the mouth of the Columbia River. Density distributions of murres estimated from ship and aerial surveys in June and July were similar to those observed in May, whereas distributions of satellite‐tagged murres in June and July indicated northward movement into British Columbia, Canada, resulting in different patterns observed from Eulerian and Lagrangian perspectives. These results suggest that the population of murres observed in the northern California Current during spring and summer includes relatively stationary individuals attending breeding colonies and nonstationary, vagile adults and subadults. Given the expected growth of telemetry studies and advances in survey technology (e.g., unmanned aerial systems), these results highlight the importance of considering methodological approaches, spatial extent, and synopticity of distribution data sets prior to integrating data from different sampling perspectives.     

Anthropometric correlates of C-reactive protein among indigenous Siberians

C-reactive protein (CRP) is an inflammatory marker, which at low-level elevations is associated with increased cardiovascular risk. Although CRP has been extensively investigated in North American and European settings, few studies have measured CRP among non-Western groups. The present study used dried whole blood spot samples to examine high-sensitivity CRP concentrations among the Yakut (Sakha) of Siberia (85 females, 56 males; 18-58 years old). Our goals were: (1) to compare Yakut CRP concentrations with other populations; (2) to investigate sex differences; and (3) to explore anthropometric correlates of CRP. Results indicate that serum equivalent CRP concentrations are similar to those from industrializing nations, lower than US and European values, and greater than Japanese concentrations. Yakut men and women display similar CRP concentrations; however, CRP was significantly higher among men after adjustment for body fat, age, and smoking. Positive associations were documented between CRP and BMI, body fat, and central adiposity.     

Membrane topology and sequence requirements for oil body targeting of oleosin

Oleosin protein is targeted to oil bodies via the endoplasmic reticulum (ER) and consists of a lipid-submerged hydrophobic (H) domain that is flanked by cytosolic hydrophilic domains. We investigated the relationship between oleosin ER topology and its subsequent ability to target to oil bodies. Oleosin variants were created to yield differing ER membrane topologies and tagged with a reporter enzyme. Localisation was assessed by fractionation after transient expression in embryonic cells. Membrane-straddled topologies with N-terminal sequence in the ER lumen and C-terminal sequence in the cytosol were unable to target to oil bodies efficiently. Similarly, a translocated topology with only ER membrane and lumenal sequence was unable to target to oil bodies efficiently. Both topology variants accumulated proportionately higher in ER microsomal fractions, demonstrating a block in transferring from ER to oil bodies. The residual oil body accumulation for the inverted topology was shown to be because of partial adoption of native ER membrane topology, using a reporter variant, which becomes inactivated by ER-mediated glycosylation. In addition, the importance of H domain sequence for oil body targeting was assessed using variants that maintain native ER topology. The central proline knot motif (PKM) has previously been shown to be critical for oil body targeting, but here the arms of the H domain flanking this motif were shown to be interchangeable with only a moderate reduction in oil body targeting. We conclude that oil body targeting of oleosin depends on a specific ER membrane topology but does not require a specific sequence in the H domain flanking arms.     

Development and Application of a Framework for Analyzing the Impacts of Urban Transportation

To adequately analyze the impacts associated with the rising use of automobiles, an assessment framework is needed that includes environment, health, economic, and sociocultural impacts. Such a framework was developed and applied to a proposed freeway-widening project in Edmonton, Canada. The assessment framework was developed using both Multi-Criteria Analysis and the Ecosystem Approach to Human Health (Ecohealth). Community participation was vital in the application of the assessment framework to this case study. Six stakeholder groups, including community members, City Councillors, and health, environment, and transportation experts, provided needed qualitative data for the assessment framework. Quantitative data were gathered from an ecological study design that associated traffic volumes with respiratory conditions in Edmonton. Community members perceptions about the impacts of the freeway widening differed from those of the expert groups in a number of areas. Environmental and health degradation was more of an issue to community members than to expert groups. Though respiratory conditions were not projected to increase by a significant amount because of the freeway widening, further analysis is necessary on other biophysical and socioeconomic impacts listed in the assessment framework. The divergence in opinion between community members and experts suggests that more communication is needed between these groups in relation to transportation planning. The Ecohealth approach ensures that community concerns are addressed in transportation planning.     

Synthesis and protein conjugation studies of vitamin K analogues

Two vitamin K analogues bearing a carboxylic acid side chain (9a and its deuterated analogue 9b) were each synthesised in six steps from commercially available menadione. Analogue 9b was conjugated to lysozyme and bovine serum albumin (BSA) using EDCI/HOBT and by prior formation of its activated succinimidyl ester 11. Quantification of the thus formed conjugates by ESMS and LC-MS revealed that the number of equivalents of the analogue used in the couplings systematically controls the number of analogues that conjugate to the protein.