Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast

Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets

Identification of RSK and TTK as Modulators of Blood Vessel Morphogenesis Using an Embryonic Stem Cell-Based Vascular Differentiation Assay

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens

Ecosystem impacts by the Ancestral Puebloans of Chaco Canyon, New Mexico, USA

The Ancestral Puebloans occupied Chaco Canyon, in what is now the southwestern USA, for more than a millennium and harvested useful timber and fuel from the trees of distant forests as well as local woodlands, especially juniper and pinyon pine. These pinyon juniper woodland products were an essential part of the resource base from Late Archaic times (3000-100 BC) to the Bonito phase (AD 800-1140) during the great florescence of Chacoan culture. During this vast expanse of time, the availability of portions of the woodland declined. We posit, based on pollen and macrobotanical remains, that the Chaco Canyon woodlands were substantially impacted during Late Archaic to Basketmaker II times (100 BC-AD 500) when agriculture became a major means of food production and the manufacture of pottery was introduced into the canyon. By the time of the Bonito phase, the local woodlands, especially the juniper component, had been decimated by centuries of continuous extraction of a slow-growing resource. The destabilizing impact resulting from recurrent woodland harvesting likely contributed to the environmental unpredictability and difficulty in procuring essential resources suffered by the Ancestral Puebloans prior to their ultimate departure from Chaco Canyon

Water uncertainty, ritual predictability and agricultural canals at Chaco Canyon, New Mexico

The Pueblo population of Chaco Canyon during the Bonito Phase (AD 800–1130) employed agricultural strategies and water-management systems to enhance food cultivation in this unpredictable environment. Scepticism concerning the timing and effectiveness of this system, however, remains common. Using optically stimulated luminescence dating of sediments and LiDAR imaging, the authors located Bonito Phase canal features at the far west end of the canyon. Additional ED-XRF and strontium isotope (87Sr/86Sr) analyses confirm the diversion of waters from multiple sources during Chaco’s occupation. The extent of this water-management system raises new questions about social organisation and the role of ritual in facilitating responses to environmental unpredictability

Soil analysis in discussions of agricultural feasibility for ancient civilizations: A critical review and reanalysis of the data and debate from Chaco Canyon, New Mexico

<div><p>Questions about how archaeological populations obtained basic food supplies are often difficult to answer. The application of specialist techniques from non-archaeological fields typically expands our knowledge base, but can be detrimental to cultural interpretations if employed incorrectly, resulting in problematic datasets and erroneous conclusions not easily caught by the recipient archaeological community. One area where this problem has failed to find resolution is Chaco Canyon, New Mexico, the center of one of the New World’s most vibrant ancient civilizations. Discussions of agricultural feasibility and its impact on local population levels at Chaco Canyon have been heavily influenced by studies of soil salinity. A number of researchers have argued that salinized soils severely limited local agricultural production, instead suggesting food was imported from distant sources, specifically the Chuska Mountains. A careful reassessment of existing salinity data as measured by electrical conductivity reveals critical errors in data conversion and presentation that have misrepresented the character of the area’s soil and its potential impact on crops. We combine all available electrical conductivity data, including our own, and apply multiple established conversion methods in order to estimate soil salinity values and evaluate their relationship to agricultural productivity potential. Our results show that Chacoan soils display the same salinity ranges and spatial variability as soils in other documented, productive fields in semi-arid areas. Additionally, the proposed large-scale importation of food from the Chuska Mountains region has serious social implications that have not been thoroughly explored. We consider these factors and conclude that the high cost and extreme inflexibility of such a system, in combination with material evidence for local agriculture within Chaco Canyon, make this scenario highly unlikely. Both the soil salinity and archaeological data suggest that there is no justification for precluding the practice of local agriculture within Chaco Canyon.</p></div

Shows the relation of key water characteristics for 113 observations between 8/6/1976 and 10/6/1983.

<p>The average for pH (7.55) and EC (0.46) are each indicated by a solid line behind each data type. Chaco discharge is shown for visual comparison of covariation between periods of increased flow and EC.</p

Figure shows the location for all known soil salinity samples in the main are of Chaco Canyon.

<p>Larger circles are to avoid providing precise location information for non-public archaeological areas. Selected profiles are presented with values for single depth samples shown next to their location. Blue circles, connected by a simple smoothed line for visual interpretation, represent estimated EC_e values in profiles. For sources that specify a depth range for specific samples, point depth is the range midpoint. For each salinity graph, the Y-Axis is Depth (cm), and the X-Axis is Estimated EC_e. Vertical lines represent varying yield decrease thresholds for maize (moving left to right): Yellow = 0%, Peach = 10%, Orange = 25%, Brown = 50%, and (when shown) Red = 100%. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198290#pone.0198290.s002" target="_blank">S1 Table</a> for raw and converted data.</p