Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies

It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.This work was supported by grants from the United States National Institutes of Health, National Science Foundation, and Welch (F1515) and Packard Foundations to EMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Urbanization does not increase “object curiosity” in vervet monkeys, but semi-urban individuals selectively explore food-related anthropogenic items

Urban environments expose animals to abundant anthropogenic materials and foods that facilitate foraging innovations in species with opportunistic diets and high behavioral flexibility. Neophilia and exploration tendency are believed to be important behavioral traits for animals thriving in urban environments. Vervet monkeys (Chlorocebus pygerythrus) are one of few primate species that have successfully adapted to urban environments, thus making them an ideal species to study these traits. Using a within-species cross-habitat approach, we compared neophilia and exploration of novel objects (jointly referred to as “object curiosity”) between semi-urban, wild, and captive monkeys to shed light on the cognitive traits facilitating urban living. To measure “object curiosity,” we exposed monkeys to various types of novel stimuli and compared their approaches and explorative behavior. Our results revealed differences in the number of approaches and explorative behavior toward novel stimuli between the habitat types considered. Captive vervet monkeys were significantly more explorative than both semi- urban and wild troops, suggesting that positive experiences with humans and lack of predation, rather than exposure to human materials per se, influence object curiosity. Across habitats, juvenile males were the most explorative age-sex class. This is likely due to males being the dispersing sex and juveniles being more motivated to learn about their environment. Additionally, we found that items potentially associated with human food, elicited stronger explorative responses in semi-urban monkeys than non-food related objects, suggesting that their motivation to explore might be driven by “anthrophilia”, that is, their experience of rewarding foraging on similar anthropogenic food sources. We conclude that varying levels of exposure to humans, predation and pre-exposure to human food packaging explain variation in “object curiosity” in our sample of vervet monkeys

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Time course imaging reveals that punctate bodies are dynamic, shown here for HeLa cells in purine-depleted medium transfected with PAICS-RFP grown in two replicate 4 hour time series.

<p>Panels (<b>A</b>) and (<b>D</b>) show the time zero condition; panels (<b>B</b>) and (<b>E</b>) show the same cells as in (<b>A</b>) and (<b>D</b>), respectively, following two hours of growth in the same medium; panels (<b>C</b>) and (<b>F</b>) show the same cells following two hours of growth after exchanging the growth medium to purine-rich. Cells marked by * display formation of punctate bodies over the time series, while cells marked by <b>p</b> display variable dynamics of punctate bodies. The # sign marks cells with punctate bodies that die over the course of the series; the cell marked by <b>d</b> dies in the absence of punctate bodies. Cell death was determined by marked cell shrinkage and membrane blebbing, detected by differential interference contrast (<b>DIC</b>) microscopy, as in panel (<b>C-DIC</b>), accompanied by markedly increased cellular fluorescence, easily distinguishable from flat healthy cells and mitotic cells (one is marked by <b>m</b> in panels (<b>C</b>) and (<b>C-DIC</b>)). Notably, punctate bodies are detectable in both purine-poor and rich media, with some forming even after the shift into purine-rich medium, as for the cell marked * in (<b>E-F</b>).</p

Formation of intracellular bodies in HEK293T cells scaled with DNA transfected.

<p>Among successfully transfected cells, the fraction of the cell population exhibiting PAICS-RFP puncta correlated strongly with the quantity of plasmid DNA transfected. Bars indicate average +/- 1 s. d. across at least 3 replicates, <i>n</i>  =  498, 627, and 591 cells, respectively.</p

Purine biosynthesis enzymes only rarely co-localized in intracellular bodies, showing even partial co-localization in no more than 4% of co-transfected cells as assayed in HeLa cells and quantified in Table S1.

<p>The top row shows an example of partial but minimal co-localization of FGAMS-EGFP and PAICS-RFP bodies. The middle row shows an example of non-colocalizing FGAMS-EGFP and ADSL-RFP bodies. The bottom row shows an example of a more typical case, non-co-localization due to the formation of bodies by only one protein in doubly-transfected cells, as shown here for PPAT-EGFP and PAICS-RFP.</p

The cell stressor hydrogen peroxide (H₂O₂) strongly induced purine biosynthetic enzyme punctate bodies regardless of hypoxanthine (Hx) presence.

<p>Base medium is DMEM supplemented with 10% FBS. As indicated, medium was also supplemented with 1 mM H₂O₂ and/or 35 µM Hx as described in Methods. For HEK293 PPAT-EGFP cells, <i>n</i> = 4419, 2652, 3088, 3182 cells per bar. For HEK293 PAICS-RFP cells, <i>n</i> = 2970, 1944, 1880, 1760. For HEK293T PPAT-EGFP cells, <i>n</i> = 4537, 2267, 2411, 2947. For HEK293T PAICS-RFP cells, <i>n</i> = 4612, 3660, 4211, 3760. Bars indicate average +/- 1 s. d. across at least 3 replicates.</p

Endogenous markers of aggregated proteins associated with intracellular foci of transfected purine biosynthetic enzymes.

<p>(<b>A-C</b>) Endogenous HSP70 and (<b>D-F</b>) ubiquitin co-localized with bodies formed in cells transfected with FGAMS-EGFP, as assayed using immunofluorescence. Immunofluorescence against endogenous glutamine synthetase (<b>G-I</b>) or GAPDH (<b>J-L</b>), which are not markers for protein aggregation, confirmed that these proteins did not co-localize with the bodies. (<b>M-O</b>) Additional control experiments employing only the secondary antibodies (tested for both secondary antibodies and shown here for Alexa Fluor 594-conjugated goat anti-rabbit) exhibited no positive signal and, with the experiments in panels (<b>G-L</b>), ruled out the possibility of non-specific antibody-mediated localization to the bodies.</p

Inhibition of the proteasome with the drug MG-132 induced formation of PPAT-EGFP bodies in a time-dependent manner.

<p>HeLa cells treated with 20 µM MG-132 in DMSO for 1.5 hours, 2.5 hours, 5 hours, and 8 hours showed increasing fractions of the cell population exhibiting PPAT-EGFP puncta. Control treatments with only the carrier (DMSO) for 8 hours showed only minimal penetrance, consistent with no treatment (<b>Fig. 1B</b>). Bars indicate average +/- 1 s. d. across 3 replicates, <i>n</i>  =  625, 793, 1024, 466, and 397 cells, respectively.</p

Chaperone activity modulated the formation of intracellular bodies of purine biosynthetic enzymes.

<p>(<b>A</b>) Short-term treatment with the HSP90 chaperone inhibitor geldanamycin induced puncta formation in a concentration-dependent manner, shown here for PPAT-EGFP in HEK293T cells. Bars indicate average +/- 1 s. d. across 3 replicates, <i>n</i>  =  680, 351, 565, 601, and 616 total cells, respectively. (<b>B</b>) Low-dosage geldanamycin pre-treatment—known to induce HSP70 activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056203#pone.0056203-McLean1" target="_blank">[11]</a>—suppressed oxidatively-induced puncta formation, shown here for PPAT-EGFP in HEK293T cells. Bars indicate average +/- 1 s. d. across 3 replicates, n  =  555, 500, 608, and 601 total cells, respectively. Results were similar for FGAMS-EGFP in the absence of hydrogen peroxide (data not shown).</p