Comparative Analysis of the Saccharomyces cerevisiae and Caenorhabditis elegans Protein Interaction Network

Protein interaction networks aim to summarize the complex interplay of proteins in an organism. Early studies suggested that the position of a protein in the network determines its evolutionary rate but there has been considerable disagreement as to what extent other factors, such as protein abundance, modify this reported dependence. We compare the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans with those of closely related species to elucidate the recent evolutionary history of their respective protein interaction networks. Interaction and expression data are studied in the light of a detailed phylogenetic analysis. The underlying network structure is incorporated explicitly into the statistical analysis. The increased phylogenetic resolution, paired with high-quality interaction data, allows us to resolve the way in which protein interaction network structure and abundance of proteins affect the evolutionary rate. We find that expression levels are better predictors of the evolutionary rate than a protein's connectivity. Detailed analysis of the two organisms also shows that the evolutionary rates of interacting proteins are not sufficiently similar to be mutually predictive. It appears that meaningful inferences about the evolution of protein interaction networks require comparative analysis of reasonably closely related species. The signature of protein evolution is shaped by a protein's abundance in the organism and its function and the biological process it is involved in. Its position in the interaction networks and its connectivity may modulate this but they appear to have only minor influence on a protein's evolutionary rate.Comment: Accepted for publication in BMC Evolutionary Biolog

Protein evolution speed depends on its stability and abundance and on chaperone concentrations.

Proteins evolve at different rates. What drives the speed of protein sequence changes? Two main factors are a protein's folding stability and aggregation propensity. By combining the hydrophobic-polar (HP) model with the Zwanzig-Szabo-Bagchi rate theory, we find that: (i) Adaptation is strongly accelerated by selection pressure, explaining the broad variation from days to thousands of years over which organisms adapt to new environments. (ii) The proteins that adapt fastest are those that are not very stably folded, because their fitness landscapes are steepest. And because heating destabilizes folded proteins, we predict that cells should adapt faster when put into warmer rather than cooler environments. (iii) Increasing protein abundance slows down evolution (the substitution rate of the sequence) because a typical protein is not perfectly fit, so increasing its number of copies reduces the cell's fitness. (iv) However, chaperones can mitigate this abundance effect and accelerate evolution (also called evolutionary capacitance) by effectively enhancing protein stability. This model explains key observations about protein evolution rates

Mechanistic target of rapamycin (mTOR) activation during ruminant mammary development and function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

This thesis examines the abundance of total and activated mechanistic target of rapamycin (mTOR) pathway components in the developing and functional ruminant mammary gland. mTOR pathway activation is stimulated by a wide range of intra- and extracellular signals, such as amino acids (AA) and hormones, making the mTOR pathway a potential candidate for the development of intervention strategies designed to increase ruminant lactation potential. Tissues from two trials shown to improve lactation potential; dam-fetal nutrition and exogenous growth hormone (GH) administration during lactation, were used to measure changes in total and activated mTOR pathway protein abundance. Results show mammary glands of d 140 fetal lambs carried by maintenance fed dams and dairy cows administered exogenous GH, had increased abundance of total and activated mTOR and mitogen activated protein kinase (MAPK) pathway proteins. Increased abundance was associated with changes in biochemical indices. In the GH study MAPK pathway activation was stimulated by IGF-1 signaling whilst mTOR pathway activation was proposed to be mediated by AA signalling. Data from the GH study shows, L-arginine a known activator of the mTOR pathway, was the only AA reduced in both plasma and the lactating gland. Upstream factors were not identified for the phenotype observed in the dam-fetal nutrition study, but similar mechanisms were proposed. To elucidate the potential regulation of mTOR pathway activation by L-arginine and examine the effect on milk production, in vitro bovine cell culture models were evaluated. Results show that none of the models evaluated produced a lactating phenotype – a pre-requisite to accurately study the lactating gland in vitro. Finally, this thesis shows L-arginine administration from d 100 to d 140 of pregnancy, in twin bearing ewes had no effect on mTOR protein abundance or activation. However, administration from d 100 to parturition improved maternal gland health. In summary, this thesis associates improved lactation potential with increased total and activated mTOR pathway protein abundance, and the administration of L-arginine during late gestation with improved gland health. These findings provide fundamental knowledge that may lead to the development of novel technologies to increase ruminant gland performance and health

Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used a new technique of site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA - an essential chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to determine how changes in protein stability, activity and abundance affect fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance; suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few strains carrying mutant DHFR proteins aggregated rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42oC by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression

Objective To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. Design Analysis of human oocytes with the use of NanoString and immunoblotting. Setting University-affiliated reproductive medicine unit. Patients Women undergoing in vitro fertilization. Intervention Human oocytes were analyzed with the use of NanoString or immunoblotting. Main Outcome Measures The abundance of transcripts for ten functionally important genes—AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2—and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. Result(s) All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes—all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy—were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. Conclusion(s) The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected

Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusions Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors

Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations.

Protein stability is widely recognized as a major evolutionary constraint. However, the relation between mutation-induced perturbations of protein stability and biological fitness has remained elusive. Here we explore this relation by introducing a selected set of mostly destabilizing mutations into an essential chromosomal gene of E.coli encoding dihydrofolate reductase (DHFR) to determine how changes in protein stability, activity and abundance affect fitness. Several mutant strains showed no growth while many exhibited fitness higher than wild type. Overexpression of chaperonins (GroEL/ES) buffered the effect of mutations by rescuing the lethal phenotypes and worsening better-fit strains. Changes in stability affect fitness by mediating the abundance of active and soluble proteins; DHFR of lethal strains aggregates, while destabilized DHFR of high fitness strains remains monomeric and soluble at 30oC and forms soluble oligomers at 42oC. These results suggest an evolutionary path where mutational destabilization is counterbalanced by specific oligomerization protecting proteins from aggregation