JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available

Pharmacokinetics of Quinacrine Efflux from Mouse Brain via the P-glycoprotein Efflux Transporter

The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp–deficient Mdr10/0 mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr10/0 brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS

Continuous Quinacrine Treatment Results in the Formation of Drug-Resistant Prions

Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy in vivo. As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR0/0 mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 µM of quinacrine in their brains without acute toxicity. PrPSc levels in the brains of prion-inoculated MDR0/0 mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrPSc levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR0/0 mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrPSc levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrPSc that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR0/0 mice. From these data, we propose that quinacrine eliminates a specific subset of PrPSc conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs

Global quantification of proteome dynamics

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2017.Living cells continuously degrade and resynthesize their constituent proteins. The maintenance of protein homeostasis is fundamental to cell survival and function. Recent advances in mass spectrometry, especially the development of stable isotope labeling with amino acids in cell culture (SILAC), have enabled proteome-wide analyses of cellular protein turnover and elucidation of protein homeostasis maintenance mechanisms. However, more efficient methods are still needed for higher precision analysis of proteome dynamics. This work first clarified one overlooked issue in the interpretation of dynamic SILAC experiments and indicated that in typical experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins. Second, a methodology that combines metabolic isotopic labeling (SILAC) with isobaric tagging (Tandem Mass Tags - TMT) was established for analysis of multiplexed samples. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. By globally quantifying the kinetics of protein clearance and synthesis, this approach provided important insights into the regulation of the proteome as fibroblasts transit from a dividing to a quiescent state. Our results indicated that, in quiescent cells, protein synthesis decreases, while protein degradation increases by up-regulation of autophagy and lysosome biogenesis. Lastly, by measuring protein degradation rates in wildtype and autophagy-deficient cells, we investigated the selectivity of macroautophagy on a global scale. Together, this work developed a more efficient methodology for measuring protein synthesis and turnover rates on a global scale and revealed an important mechanism of protein homeostasis in quiescent fibroblasts

Global Analysis of Cellular Protein Flux Quantifies the Selectivity of Basal Autophagy

In eukaryotic cells, macroautophagy is a catabolic pathway implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins has not been quantified on proteome-wide scales. In this study, we created autophagy-deficient primary human fibroblasts and quantified the resulting changes in basal degradative flux by dynamic proteomics. Our results provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. The data indicate that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome at varying levels. As contrasting examples, we demonstrate that the proteasome and CCT/TRiC chaperonin are robust substrates of basal autophagy, whereas the ribosome is largely protected under basal conditions. This selectivity may establish a proteostatic feedback mechanism that stabilizes the proteasome and CCT/TRiC when autophagy is inhibited

Turnover and replication analysis by isotope labeling (TRAIL) reveals the influence of tissue context on protein and organelle lifetimes

Abstract The lifespans of proteins range from minutes to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long‐lived proteins accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell turnover and proteolytic degradation contribute to protein turnover. We develop turnover and replication analysis by 15N isotope labeling (TRAIL) to quantify protein and cell lifetimes with high precision and demonstrate that cell turnover, sequence‐encoded features, and environmental factors modulate protein lifespan across tissues. Cell and protein turnover flux are comparable in proliferative tissues, while protein turnover outpaces cell turnover in slowly proliferative tissues. Physicochemical features such as hydrophobicity, charge, and disorder influence protein turnover in slowly proliferative tissues, but protein turnover is much less sequence‐selective in highly proliferative tissues. Protein lifetimes vary nonrandomly across tissues after correcting for cell turnover. Multiprotein complexes such as the ribosome have consistent lifetimes across tissues, while mitochondria, peroxisomes, and lipid droplets have variable lifetimes. TRAIL can be used to explore how environment, aging, and disease affect tissue homeostasis