A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.

Resurrecting ancestral alcohol dehydrogenases from yeast.

Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used Adh(A) to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

Amino acid homeostasis and chronological longevity in Saccharomyces cerevisiae.

Understanding how non-dividing cells remain viable over long periods of time, which may be decades in humans, is of central importance in understanding mechanisms of aging and longevity. The long-term viability of non-dividing cells, known as chronological longevity, relies on cellular processes that degrade old components and replace them with new ones. Key among these processes is amino acid homeostasis. Amino acid homeostasis requires three principal functions: amino acid uptake, de novo synthesis, and recycling. Autophagy plays a key role in recycling amino acids and other metabolic building blocks, while at the same time removing damaged cellular components such as mitochondria and other organelles. Regulation of amino acid homeostasis and autophagy is accomplished by a complex web of pathways that interact because of the functional overlap at the level of recycling. It is becoming increasingly clear that amino acid homeostasis and autophagy play important roles in chronological longevity in yeast and higher organisms. Our goal in this chapter is to focus on mechanisms and pathways that link amino acid homeostasis, autophagy, and chronological longevity in yeast, and explore their relevance to aging and longevity in higher eukaryotes.

New insights into the role of mitochondria in aging: mitochondrial dynamics and more.

A decline in mitochondrial function plays a key role in the aging process and increases the incidence of age-related disorders. A deeper understanding of the intricate nature of mitochondrial dynamics, which is described as the balance between mitochondrial fusion and fission, has revealed that functional and structural alterations in mitochondrial morphology are important factors in several key pathologies associated with aging. Indeed, a recent wave of studies has demonstrated the pleiotropic role of fusion and fission proteins in numerous cellular processes, including mitochondrial metabolism, redox signaling, the maintenance of mitochondrial DNA and cell death. Additionally, mitochondrial fusion and fission, together with autophagy, have been proposed to form a quality-maintenance mechanism that facilitates the removal of damaged mitochondria from the cell, a process that is particularly important to forestall aging. Thus, dysfunctional regulation of mitochondrial dynamics might be one of the intrinsic causes of mitochondrial dysfunction, which contributes to oxidative stress and cell death during the aging process. In this Commentary, we discuss recent studies that have converged at a consensus regarding the involvement of mitochondrial dynamics in key cellular processes, and introduce a possible link between abnormal mitochondrial dynamics and aging.

Acetyl-coenzyme A synthetase 2 is a nuclear protein required for replicative longevity in Saccharomyces cerevisiae.

Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2 Delta strain has a reduced replicative life span compared to wild-type and acs1 Delta strains. Furthermore, replicatively aged acs2 Delta cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2 Delta strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast.

Dosimetric considerations of 99mTc-MDP uptake within the epiphyseal plates of the long bones of pediatric patients.

Skeletal scintigraphy is most performed in pediatric patients using the radiopharmaceutical 99mTc labelled methylene diphosphonate (99mTc-MDP). Reference biokinetic models for 99mTc-MDP indicate 50% of the administered activity is uniformly localized to the interior bone surfaces (trabecular and cortical regions), yet imaging data clearly show some preferential uptake to the epiphyseal growth plates of the long bones. To explore the dosimetric consequences of these regional activity concentrations, we have modified mesh-type computational phantoms of the International Commission on Radiological Protection (ICRP) reference pediatric series to explicitly include geometric models of the epiphyseal growth plates (2 mm in thickness) within the left/right, distal/proximal ends of the humeri, radii, ulnae, femora, tibia, and fibulae. Bone mineral activity from the ICRP Publication 128 biokinetic model for 99mTc-MDP (ICRP 2015) was then partitioned to the growth plates at values of 0.5%, 4.4%, 8.3%, 12.2%, 16.1%, and 20%. Radiation transport simulations were performed to compute 99mTc S-values and organ dose coefficients to the soft tissues and to bone site-specific regions of spongiosa. As the percentage of bone activity assigned to the growth plates was increased (from 0.5% to 20%), absorbed doses to the soft tissue organs, active bone marrow, bone endosteum (BE), as well as the detriment-weighted dose, were shown to decrease from their nominal values (no substantial growth plate activity), while epiphyseal plate self-doses increased. In the 15 year old male phantom, moving from 0.5% to 20% relative bone activity within the epiphyseal plates resulted in a 15% reduction in active marrow (AM) and BE dose, a 10% reduction in mean soft tissue and detriment-weighted dose, and a 6.3-fold increase in epiphyseal plate self-dose. In the newborn female phantom, we observed a 18% decrease in AM and BE dose, a 10% decrease in mean soft tissue dose, a 15% decrease in detriment-weighted dose, and 12.8-fold increase in epiphyseal plate self-dose. Increases (to 3 mm) and decreases (to 1 mm) in the assumed growth plate thickness of our models were shown to impact only the growth plate self-dose. Future work in differential quantification of 99mTc-MDP activity-growth plates versus other bone surfaces-is required to provide clinically realistic data on activity partitioning as a function of patient age, and perhaps skeletal site. The phantom series presented here may be used to develop more optimized age-related guidance on 99mTc-MDP administered activities to children.

Role of histone deacetylase Rpd3 in regulating rRNA gene transcription and nucleolar structure in yeast.

The 35S rRNA genes at the RDN1 locus in Saccharomyces cerevisiae can be transcribed by RNA polymerase (Pol) II in addition to Pol I, but Pol II transcription is usually silenced. The deletion of RRN9 encoding an essential subunit of the Pol I transcription factor, upstream activation factor, is known to abolish Pol I transcription and derepress Pol II transcription of rRNA genes, giving rise to polymerase switched (PSW) variants. We found that deletion of histone deacetylase gene RPD3 inhibits the appearance of PSW variants in rrn9 deletion mutants. This inhibition can be explained by the observed specific inhibition of Pol II transcription of rRNA genes by the rpd3Delta mutation. We propose that Rpd3 plays a role in the maintenance of an rRNA gene chromatin structure(s) that allows Pol II transcription of rRNA genes, which may explain the apparently paradoxical previous observation that rpd3 mutations increase, rather than decrease, silencing of reporter Pol II genes inserted in rRNA genes. We have additionally demonstrated that Rpd3 is not required for inhibition of Pol I transcription by rapamycin, supporting the model that Tor-dependent repression of the active form of rRNA genes during entry into stationary phase is Rpd3 independent.

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus.

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.

2-micron circle plasmids do not reduce yeast life span.

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir+ and cir0 strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.

Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast.

We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

The UF family of hybrid phantoms of the developing human fetus for computational radiation dosimetry.

Historically, the development of computational phantoms for radiation dosimetry has primarily been directed at capturing and representing adult and pediatric anatomy, with less emphasis devoted to models of the human fetus. As concern grows over possible radiation-induced cancers from medical and non-medical exposures of the pregnant female, the need to better quantify fetal radiation doses, particularly at the organ-level, also increases. Studies such as the European Union's SOLO (Epidemiological Studies of Exposed Southern Urals Populations) hope to improve our understanding of cancer risks following chronic in utero radiation exposure. For projects such as SOLO, currently available fetal anatomic models do not provide sufficient anatomical detail for organ-level dose assessment. To address this need, two fetal hybrid computational phantoms were constructed using high-quality magnetic resonance imaging and computed tomography image sets obtained for two well-preserved fetal specimens aged 11.5 and 21 weeks post-conception. Individual soft tissue organs, bone sites and outer body contours were segmented from these images using 3D-DOCTOR™ and then imported to the 3D modeling software package Rhinoceros™ for further modeling and conversion of soft tissue organs, certain bone sites and outer body contours to deformable non-uniform rational B-spline surfaces. The two specimen-specific phantoms, along with a modified version of the 38 week UF hybrid newborn phantom, comprised a set of base phantoms from which a series of hybrid computational phantoms was derived for fetal ages 8, 10, 15, 20, 25, 30, 35 and 38 weeks post-conception. The methodology used to construct the series of phantoms accounted for the following age-dependent parameters: (1) variations in skeletal size and proportion, (2) bone-dependent variations in relative levels of bone growth, (3) variations in individual organ masses and total fetal masses and (4) statistical percentile variations in skeletal size, individual organ masses and total fetal masses. The resulting series of fetal hybrid computational phantoms is applicable to organ-level and bone-level internal and external radiation dosimetry for human fetuses of various ages and weight percentiles.

Gingival RAGE expression in calorie-restricted versus ad libitum-fed rats.

BACKGROUND: The receptor for advanced glycation end products (RAGE) is a multiligand receptor expressed in a number of cell types, including gingival epithelia. RAGE mediates inflammation and induces cellular oxidative stress. Upregulation of RAGE is associated with various diseases, such as periodontal and cardiovascular diseases. This study examines the hypothesis that the gingiva of rats fed a calorie-restriction (CR) diet expresses lower levels of RAGE than the gingiva of rats fed an ad libitum (AL) diet. METHODS: Male F344BN rats (n = 16) from the National Institute on Aging (NIA) were fed a CR (n = 8) or AL (n = 8) diet according to NIA recommendations. Rats were sacrificed by guillotine at 8 (n = 5), 18 (n = 3), 29 (n = 4), and 38 (n = 4) months of age. The gingiva from around the molars was dissected and submitted for histologic and molecular analyses. RESULTS: Immunohistochemistry revealed that RAGE was expressed in the plasma membrane and cytoplasm of gingival epithelial cells and endothelial cells from both groups. RAGE mRNA levels were quantified relative to levels of GAPDH mRNA by real-time reverse-transcriptase polymerase chain reaction. The mean relative RAGE mRNA level in the CR group (7.26 ± 0.54) was lower than in the AL group (10 ± 1.4) (P <0.05). There were no detectable differences in RAGE expression according to animal age. CONCLUSION: Gingival RAGE expression in rats is reduced by calorie restriction.

Autophagy is required for extension of yeast chronological life span by rapamycin.

Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.

Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiae.

Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast.

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit.

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in "half-mer" polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Delta cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Delta cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Delta cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.

Plasmid accumulation reduces life span in Saccharomyces cerevisiae.

Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.