Yeast Tdh3 (glyceraldehyde 3-phosphate dehydrogenase) is a Sir2-interacting factor that regulates transcriptional silencing and rDNA recombination

Sir2 is an NAD(+)-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD(+)-binding protein, influences nuclear NAD(+) levels; we speculate that Tdh3 links nuclear Sir2 with NAD(+) from the cytoplasm

Combined Vorinostat and Chloroquine Inhibit Sodium Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo

Purpose Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo.Experimental DesignInformed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice.ResultsWe identify an acidic dipeptide within the NIS C-terminus which mediates binding to the 2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/ SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence.ConclusionsNIS internalisation is specifically druggable in vivo. Our data therefore provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer.<br/

Burden of Gastrointestinal Disease in the United States: 2012 Update

Gastrointestinal (GI) diseases account for substantial morbidity, mortality and cost. Statistical analyses of the most recent data are necessary to guide GI research, education and clinical practice. We estimate the burden of GI disease in the US

Development of screening questions for doctor–patient consultation assessing the quality of life and psychosocial burden of glioma patients: an explorative study

Purpose: Psychosocial screening for glioma patients is challenging because many patients suffer from neurocognitive deficits, which may impair assessment. This study's aim was to exploratively develop three screening questions for unmet needs to prospectively be applicable in patient-doctor consultation. Methods: Patient interviews, a survey for health-care professionals and a weighted scoring procedure were developed for this study. Six main areas were defined according to main areas of validated questionnaires (psyche, cognition, body, role functioning, social support, unmet needs). Patients and health-care professionals rated the importance of these areas and corresponding items, patients additionally stated whether the issues addressed affected them. Results: A total of 50 patients were included, and 36 health-care professionals participated in the online survey. The three areas (psyche, body and cognition) considered to be most relevant by both, health-care professionals and patients, generated three screening questions. If the patient was affected by the issue addressed with a screening question, a subordinate question from that area that our patient sample considered most important could additionally be asked. The elaborated screening questions are the following: (1) main area psyche: 'Has your mood worsened?', (2) main area body: 'Do physical changes put a strain on you?', and (3) main area cognition: 'Has your memory capacity worsened?' Conclusion: These questions represent a basis for further research regarding their application in neuro-oncological clinical routine

Separation of silencing and GAPDH activity in <i>TDH3</i> alleles.

<p>(A) <i>TDH3</i> mutants influence transcriptional silencing at yeast telomeres. For each allele the wild-type amino acid and position is noted, followed by the amino acid replacing it in the mutated allele. A phenotypic assay measuring silencing of a <i>URA3</i> reporter gene was conducted as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen-1003871-g001" target="_blank">Figure 1</a> legend. (B) mRNA levels of <i>YFR057W</i>, a naturally occurring telomere proximal gene, were determined as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen-1003871-g001" target="_blank">Figure 1</a> legend. (C) GAPDH levels of strains bearing <i>TDH3</i> alleles. Levels of glyceraldehyde phosphate dehydrogenase activity were measured in extracts made from the indicated strains, as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Ralser3" target="_blank">[64]</a>.</p

Physical and functional interaction between Tdh3 and Sir2 in a two-hybrid assay.

<p>(A) <i>TDH3</i> fused to the DNA-binding domain results in the repression of the <i>HIS3</i> reporter gene. Two-hybrid assays were performed as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-James1" target="_blank">[69]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Uetz1" target="_blank">[70]</a> using the complete Sir2 and Tdh3 open reading frames. Rows are labeled with the activation-domain fusions used; pOAD is the vector control. Each column lists the binding-domain fusion used; pOBD is the vector control. Tdh3ΔBD indicates strains that overexpress <i>TDH3</i> from the pOBD vector lacking the Gal4 binding domain. (B) Elevated Tdh3 increases Sir2-dependent repression of a reporter gene. Labels are as described in (A). Sir4Δ730N-AD was included as a positive control for Sir2 interaction. (C) Tdh3 and Sir2 interact in vivo. The activation domain and binding domain fusions from (A) and (B) were assessed in a strain lacking the <i>SIR2, SIR3, and SIR4</i> genes (YSH625).</p

Tdh3 is a novel regulator of Sir2 dependent transcriptional silencing.

<p>(A) Tdh3 regulates silencing at the telomeres. Serial dilutions of strains bearing <i>URA3</i> reporter gene adjacent to a telomere <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Roy1" target="_blank">[17]</a> were made on complete medium (SDC), and on media containing 5-FOA, which counterselects for <i>URA3</i> expression. The <i>URA3</i> promoter is approximately 1 kb from the telomere repeat sequences <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Renauld1" target="_blank">[67]</a>. (B) Overexpression of Tdh3 causes an increase in silencing at the <i>HMR</i> locus. A plasmid containing the <i>TDH3</i> gene fused to the galactose-inducible <i>GAL1</i> promoter was introduced into a strain bearing the <i>ADE2</i> gene at the <i>HMR</i> locus <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Sussel1" target="_blank">[68]</a>. This strain lacks the Orc binding site at the <i>HMR-E</i> silencer (the “A site” of the silencer). Serial dilutions of this stain were grown on the indicated media. (C) Tdh3 regulates expression of an endogenous telomere proximal gene. Expression of the native telomere gene <i>YFR057W</i>, was examined by quantitative RT-PCR <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-MartinsTaylor1" target="_blank">[60]</a> in the indicated strains. (D) Sir2 protein levels are unchanged in strains lacking or overexpressing Tdh3. Left panel: Sir2 expressed from its endogenous gene was detected via immunoblotting protein extracts made from a wild-type strain, a strain lacking the <i>TDH3</i> gene, or a strain expressing a Tdh3 protein with a single amino acid substitution. Right panel: Strains overexpressing Sir2 and bearing either a control vector (pRS416) or a plasmid overexpressing Tdh3 are shown.</p

Tdh3 affects nuclear NAD⁺ levels in yeast.

<p>(A) <i>TDH3</i> deletion or overexpression does not affect overall cellular NAD⁺ levels. Left panel: relative NAD⁺ levels are shown for strains lacking the <i>TDH3</i> or <i>NPT1</i> genes, and their matched wild-type strains. Right panel: relative NAD⁺ levels are shown in a strain overexpressing the <i>TDH3</i> gene and in a vector control strain. (B) Tdh3 maintains nuclear NAD⁺ levels. Nuclear NAD⁺ was measured using an NAD<b>⁺</b>-sensitive transcriptional reporter gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Anderson1" target="_blank">[31]</a>. Strains expressed the NAD<b>⁺</b>-dependent transcriptional activator from a <i>LEU2</i>-marked plasmid. Control strains lacked the binding site for the transcriptional activator (no NAD box) or lacked the activator (no NadR-Gal4AD). Serial dilutions of the listed strains were plated on the indicated media. Levels of the NadR-Gal4AD protein were similar in wild type and <i>Δtdh3</i> cells (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871.s003" target="_blank">Figure S3</a>). (C) Tdh3 and Npt1 have a redundant role in promoting cell growth. Doubling times of the indicated single and double mutant strains is shown. (D) Silencing at the telomere in <i>Δnpt1 Δtdh3</i> strains. Expression of the native telomere gene <i>YFR057W</i>, was examined by quantitative RT-PCR in the indicated strains.</p

Strains.

<p>Strains.</p