Brian Wasko
Permanent URI for this collectionhttps://hdl.handle.net/10657.1/2515
Dr. Brian Wasko is an Assistant Professor at University of Houston-Clear Lake. Dr. Wasko's research interests include Biology of Aging, Cellular pH and Metal Homeostasis, Basic Molecular and Cellular Biology.
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Item Bisphosphonates Induce Autophagy by Depleting Geranylgeranyl Diphosphate(J Pharmacol Exp Ther., 2011) Wasko, BrianMultiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates in the induction of autophagy. However, the exact mechanism by which isoprenoid biosynthesis inhibitors induce autophagy has not been well established. We hypothesized that inhibition of farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGDPS) by bisphosphonates would induce autophagy by depleting cellular geranylgeranyl diphosphate (GGPP) and impairing protein geranylgeranylation. Herein, we show that an inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by the accumulation of the autophagic marker LC3-II. Treatment of cells with lysosomal protease inhibitors [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and pepstatin A] in combination with zoledronate or digeranyl bisphosphate further enhances the formation of LC3-II, indicating that these compounds induce autophagic flux. It is noteworthy that the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) geranylgeranylation by isoprenoid pathway inhibitors (lovastatin), zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase 1 substrate) geranylgeranylation. In addition, specific inhibitors of farnesyl transferase and geranylgeranyl transferase I are unable to induce autophagy in our system. Furthermore, the addition of bafilomycin A1 (an inhibitor of autophagy processing) enhancer the antiproliferative effects of digeranyl bisphosphonate. These results are the first to demonstrate that bisphosphonates induce autophagy. Our study suggests that induction of autophagy in PC3 cells with these agents is probably dependent upon impairment of geranylgeranylation of GGTase II substrates.Item Buffering the pH of the Culture Medium does not Extend Yeast Replicative Lifespan(F1000 Research, 2013) Wasko, BrianDuring chronological aging of the budding yeast cells, the culture medium can become acidified and this acidification limits cell survival. As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium. In this study, we assessed whether a similar process occur during replicative aging of yeast cells. We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan. Thus we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.Item CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes(PNAS, 2018) Wasko, BrianTranscriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but has been relatively under studied in this context. Here we used RNA-seq and ri-bosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity.Item CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes(Cell Rep, 2017) Wasko, BrianTranscriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but has been relatively under studied in this context. Here we used RNA-seq and ri-bosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity.Item A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging(Cell Metab, 2015) Wasko, BrianMany genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is non-additive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.Item Defining Molecular Basis for the Longevity Traits in Natural Yeast Isolates(NPJ Aging Mechanisms of Disease, 2015) Wasko, BrianBackground: The budding yeast has served as a useful model organism in aging studies, leading to the identification of genetic determinants of longevity, many of which are conserved in higher eukaryotes. However, factors that promote longevity in a laboratory setting often have severe fitness disadvantages in the wild. Aims and Methods: To obtain an unbiased view on longevity regulation, we analyzed how a replicative lifespan is shaped by transcriptional, translational, metabolic, and morphological factors across 22 wild-type Saccharomyces cerevisiae isolates. Results: We observed significant differences in lifespan across these strains and found that their longevity is strongly associated with up-regulation of oxidative phosphorylation and respiration and down-regulation of amino- acid and nitrogen compound biosynthesis. Conclusions: As calorie restriction and TOR signaling also extend the lifespan by adjusting many of the identified pathways, the data suggest that the natural plasticity of yeast lifespan is shaped by the processes that not only do not impose cost on fitness, but also are amenable to dietary interventionItem Defining the Impact of Mutation Accumulation on Replicative Lifespan in Yeast Using Cancer-associated Mutator Phenotypes(PNAS, 2019) Wasko, BrianMutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Humans cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polℇ) or lagging strand (Polō) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decrease cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burdensItem Dietary restriction and mitochondrial function link replicative and chronological aging in Saccharomyces cerevisiae(Experimental Gerontology, 2012) Wasko, BrianChronological aging of budding yeast cells result in a reduction in a subsequent replicative life span through unknown mechanisms. Here, we show that dietary restrictions during chronological aging delays the reduction in subsequent replicative life span up to at least 23 days of chronological age. We further show that among the viable portion of the control population aged 26 days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role of mitochondrial function in this process.Item End-of-lfe Cell Cycle Arrest Contributes to Stochasticity of Yeast Replicative Aging(FEMS Yeast Research, 2013) Wasko, BrianThere is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs 35.6) and larger coefficient of variance with respect to individual life spans (0,42 vs 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.Item Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae(DNA Repair, 2009) Wasko, BrianYeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5’ end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was coinactivated. Defective recombination repair of DSBs in mrx cells thus arises from at least two separate processes: loss of mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.Item Loss of Vacuolar Acidity Results in Iron Sulfur Cluster Defects and Divergent Homeostatic Responses During Aging in Saccharomyces Cerevisiae(GeroScience, 2020) Wasko, BrianThe loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determine that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homeogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contract, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.Item Molecular Mechanisms Underlying Genotype-dependent Responses to Dietary Restrictions(Aging Cell, 2013) Wasko, BrianDietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effects of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of Dr on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae, Resulting changes in mean lifespan ranged from reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processed that underlie genotype-dependent effects of DR that may be important modulations of DR in high organisms.Item mTOR Inhibition by Rapamycin Alleviates Mitochondrial Disease in the Ndufs4-Mouse Model of Leigh Syndrome(Science, 2013) Wasko, BrianMitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufsf [NADH dehydrogenase (ubiquinone) FE-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.Item Nar1 Deficiency Results in Shortened Lifespan and Sensitivity to Paraquat that is Rescued by Increased Expression of Mitochondrial Superoxide Dismutase(Mech Ageing Dev, 2014) Wasko, BrianSaccaromyces cerevisiae Nar lp is an essential Fe/S protein that exhibits striking similarity to bacterial iron-only hydogenases. Nar1p is required for the maturation of cytosolic and nuclear, but not of mitochondrial Fe/S proteins, and plays a role in modulating sensitivity to oxygen in both yeast and Caenorhabditis elegans through unknown mechanisms. Here we report that Nar1 deficiency results in shortened lifespan and sensitivity to paraquat that is rescued by increased expression of mitochondrial superoxide dismutase. These data suggest that Nar1p promotes protection against oxidative stress and define a new role for Nar1p in promoting replicative lifespan.Item New Functional and Biophysical Insights into the Mitochondrial Rieske Iron-Sulfur Protein from Genetic Suppressor Analysis(WORM, 2016) Wasko, BrianSeveral intragenic mutations suppress the C. elegans isp-1 (qm150) allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the “tether” span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with the qm150 allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a “spring-loaded” mechanism involving a binding force for formation of enzyme-substate complex, counterbalanced by forces (a chemical “spring”) favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the “spring.” In this commentary we discuss additional control features and relate the primary inhibitions to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen in isp-1 (qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity in C. elegans from the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a “canary” function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells.Item A Novel Squalene Synthase inhibitor Combined with a Statin or Nitrogenous Biphosphonate in vitro(J. Lipid Res., 2011) Wasko, BrianStatins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel biphosphonates as potent and specific inhibitors of SQS, including tetrasodium salt of 9-biphenyl-4, dimethyl-nona-3, 7-dienyl-1, 1-biphosphonic acid (Compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limited their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletionlItem pH Neutralization Protects Against Reduction in Replicative Lifespan Following Chronological Aging in Yeast(Cell Cycle, 2012) Wasko, BrianChronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standards conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronological aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.Item Pivaloyloxtmethyl-modified isoprenoid bisphosphonates display enhanced inhibition of cellular geranylgeranylation(Biorg Med Chem, 2008) Wasko, BrianNitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular geranylgeranylation even though this modification decreases their in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthaseItem PMT1 Deficiency Enhances Basal UPR Activity and Extends Replicative Lifespan of Sacchraromyces cerevisiae(Age (Dordr), 2015) Wasko, BrianPmt1p is an important member of the protein O-mannosyltransferase (PMT) family of enzymes, which participates in the endoplasmic reticulum (ER) unfolded protein response (UPR), an important pathway for alleviating ER stress. ER stress and the UPR have been implicated in aging and age-related diseases in several organisms; however, a possible role for PMT1 in determining lifespan has not been previously described. In this study, we report that deletion of PMT1 increases replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae, while overexpression of PMT1 (PMT1-OX) reduces RLS. Relative to wild-type and PMT1-OX strains, the pmt1Δ strain had enhanced HAC1 mRNA splicing and elevated expression levels of UPR target genes. Furthermore, the increase RLS of the pmt1Δ strain could be completely abolished by deletion of either IRE1 or HAC1, two upstream modulators of the UPR. The double deletion strains pmt1Δhac1Δ and pmt1Δire1Δ also displayed generally reduced transcription of UPR target genes. Collectively, our results suggest that PMT1 deficiency enhances basal activity of the ER UPR and extends the RLS of yeast mother cells through a mechanism that requires IRE1 and HAC1.Item Quantitative determination of isopentenyl diphosphate in cultured mammalian cells(Analytical Biochemistry, 2012) Wasko, BrianIsopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutarly coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas, zoledronic acid (10 uM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells.