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

On generic protein aggregation and its aging and evolutionary implications

textMany neuro-degenerative and metabolic diseases like Parkinson’s and Alzheimer’s are attributed to the effect of mis-folded and aggregated state of proteins in cells. This suggests that the phenomenon of in vivo protein aggregation may be relatively common, perhaps more than currently appreciated. In this study, we aimed to decipher the cause behind an intriguing and potentially related phenomenon observed in yeast cells - a widespread reorganization of hundreds of cytosolic proteins into punctate foci under starvation conditions. The key question that emerges is whether this phenomenon represents organization of proteins into functional assemblies or catastrophic aggregation. This thesis supports the aggregation hypothesis and provides evidence of its role in shaping the dynamics of cellular proteomes. We have been able to demonstrate that the proteins forming foci share a high propensity to aggregate and that these foci may represent sites of homogenous protein aggregation, structures which are typically associated with chaperones. A link between the formation of foci to the yeast aging process has also been established. With evidence correlating protein aggregation propensities to the cellular energy state, we have extended the current "living on the edge" hypothesis (which demonstrates an inverse correlation between protein expression levels and their aggregation propensities). For a specific case of the "purinosome", which is inferred to be a functional enzyme complex responsible for purine biosynthesis, we have shown that the observations may be explained alternatively as a generic protein aggregation phenomenon. This study highlights a systems approach to studying cellular proteins, which can corroborate or provide an alternative explanation to inferences drawn from traditional reductionistic analysis.Cellular and Molecular Biolog

Proceedings of the 34th Hawaii International Conference on System Sciences- 2001 Pattern Extraction for Monitoring Medical Practices

We present in this paper the extraction of relational patterns on a hospital discharge database to monitor for quality of care. We delineate the requirements set for the extraction of meaningful medical practices from the data and describe how relational patterns address these requirements. Patterns representing practices regarding hospitalization instances are comprehensive and easy to interpret-- making them useful for quality management decision making. We demonstrate how relational patterns can be applied to identify poor practices embedded in hospitalization processes and trigger subsequent inquiries. 1

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

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

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

Transfected cells exhibiting intracellular bodies died at a significantly greater rate than transfected cells lacking such bodies, shown here for HeLa cells two hours after either no treatment or after exchanging the growth medium from purine-depleted to purine-rich.

<p>All comparisons between cells with and without intracellular bodies are statistically significant (<i>p</i>-values ranging from 10^-4 to <10^-16). Cell death was measured as marked cell shrinkage and membrane blebbing accompanied by markedly increased cellular fluorescence during time lapse fluorescence microscopy (e.g., as for the example cells in <b>Fig. 9</b>).</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