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

Disorder, Promiscuity, and Toxic Partnerships

Many genes are toxic when overexpressed, but general mechanisms for this toxicity have proven elusive. Vavouri et al. (2009) find that intrinsic protein disorder and promiscuous molecular interactions are strong determinants of dosage sensitivity, explaining in part the toxicity of dosage-sensitive oncogenes in mice and humans

Formation of intracellular glutamine synthetase bodies depends strongly upon cellular age and glucose availability

The enzyme glutamine synthetase serves key roles in central nitrogen metabolism, catalyzing the biosynthesis of glutamine, as well as regulating ammonia assimilation and integrating metabolic signals to balance nitrogen use. The budding yeast enzyme was recently found to form intracellular bodies (GS bodies) composed of glutamine synthetase and Hsp90 chaperones following various types of nutrient depletion or chemical stress. In order to better quantify and characterize the in vivo formation of GS bodies, we developed an assay for their formation in single yeast cells using imaging flow cytometry, which enables the quantitative measurement of rates of GS body formation and their population penetrance. Either reduction of supplied glucose, or addition of the competitive inhibitor of glycolysis, 2-deoxyglucose, markedly enhanced the formation of GS bodies. The occurrence of GS bodies increased with increasing cell size, a proxy for cell age, while treatment with rapamycin antagonized their formation. Direct measurement of GS body formation as a function of replicative age showed that mother cells exhibited a significantly higher incidence of GS bodies than daughter cells, and the frequency of GS body formation increased with increasing replicative cell age. Thus, we find that yeast glutamine synthetase bodies form in a manner strongly dependent on available glucose and increase markedly with cell age.</jats:p

Co-expressed HSP70 and HSP90 chaperones marked purine biosynthesis bodies.

<p>Intracellular bodies formed by (<b>A</b>) PPAT-EGFP, (<b>D</b>) FGAMS-EGFP and (<b>G</b>) PAICS-RFP co-localized with co-transfected (<b>B,E</b>) HSP70-RFP or (<b>H</b>) HSP70-GFP, shown here in HeLa cells. (<b>C, F</b> and <b>I</b>) show merged images. (<b>J-L</b>) Intracellular bodies were also often observed to co-localize with co-transfected HSP90, shown here with PPAT-EGFP.</p

The formation of intracellular foci by transfected purine biosynthetic enzymes correlated well with the aggregation potential of the proteins but was not strongly influenced by purine availability.

<p>(<b>A</b>) The frequency with which intracellular bodies appeared across the population of transfected cells varied for individual <i>de novo</i> purine biosynthetic enzymes, and did not depend upon the purine content of the cell growth medium, shown here for HEK293 cells cultured using the optimized medium formulations as described in the Methods. Unless otherwise specified, for HEK293 cells assays in this and later figures, 200 ng of DNA were transfected for each construct. Bars in all experiments represent the average and +/- 1 s.d. from at least 3 replicates, counting <i>n</i>  =  769, 742, 755, 665, 1225, and 1016 cells, respectively. See Methods for abbreviations of protein names. (<b>B</b>) This trend was similar for HeLa cells cultured in purine-rich medium versus purine-depleted medium, formulated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056203#pone.0056203-An1" target="_blank">[6]</a>. Unless otherwise specified, for HeLa cell assays in this and later figures, 1.6 µg of DNA were transfected for each construct. Bars in all experiments represent the average and +/- 1 s.d. from at least 3 replicates, here counting <i>n</i>  =  578, 821, 662, 832, 1519, 732, 674, and 791 total cells, respectively. (<b>C</b>) The frequency with which transfected cells exhibited bodies (shown here for values reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056203#pone.0056203-An1" target="_blank">[6]</a>) correlated strongly with each protein’s predicted aggregation potential <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056203#pone.0056203-FernandezEscamilla1" target="_blank">[10]</a>. For comparison, the unrelated enzyme GAPDH has a TANGO score of 879.</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