Is Overoxidation of Peroxiredoxin Physiologically Significant?

Eukaryotic peroxiredoxins are highly susceptible to sulfinic acid formation. This overoxidation, which is thought to convert peroxiredoxins into chaperones, can be reversed by sulfiredoxins. Several organisms, including Caenorhabditis elegans, lack sulfiredoxins but encode sestrins, proteins proposed to be functionally equivalent. We induced peroxiredoxin overoxidation in C. elegans with a short peroxide pulse. We found that reduction of overoxidized peroxiredoxin 2 (PRDX-2) was extremely slow and sestrin-independent, strongly implying that worms lack an efficient repair system. Analysis of PRDX-2's overoxidation status during C. elegans lifespan revealed no accumulation of overoxidized PRDX-2 at any point, questioning whether PRDX-2 overoxidation in worms is physiologically relevant. Antioxid. Redox Signal. 14, 725-730.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90505/1/ars-2E2010-2E3717.pd

Effects of Oxidative Stress on Behavior, Physiology, and the Redox Thiol Proteome of Caenorhabditis elegans

Accumulation of reactive oxygen species has been implicated in various diseases and aging. However, the precise physiological effects of accumulating oxidants are still largely undefined. Here, we applied a short-term peroxide stress treatment to young Caenorhabditis elegans and measured behavioral, physiological, and cellular consequences. We discovered that exposure to peroxide stress causes a number of immediate changes, including loss in mobility, decreased growth rate, and decreased cellular adenosine triphosphate levels. Many of these alterations, which are highly reminiscent of changes in aging animals, are reversible, suggesting the presence of effective antioxidant systems in young C. elegans. One of these antioxidant systems involves the highly abundant protein peroxiredoxin 2 (PRDX-2), whose gene deletion causes phenotypes symptomatic of chronic peroxide stress and shortens lifespan. Applying the quantitative redox proteomic technique OxICAT to oxidatively stressed wild-type and prdx-2 deletion worms, we identified oxidation-sensitive cysteines in 40 different proteins, including proteins involved in mobility and feeding (e.g., MYO-2 and LET-75), protein translation and homeostasis (e.g., elongation factor 1 [EFT-1] and heat shock protein 1), and adenosine triphosphate regeneration (e.g., nucleoside diphosphate kinase). The oxidative modification of some of these redox-sensitive cysteines may contribute to the physiological and behavioral changes observed in oxidatively stressed animals. Antioxid. Redox Signal. 14, 1023-1037.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90439/1/ars-2E2010-2E3203.pd

Proteome analysis of monocytes implicates altered mitochondrial biology in adults reporting adverse childhood experiences.

The experience of adversity in childhood has been associated with poor health outcomes in adulthood. In search of the biological mechanisms underlying these effects, research so far focused on alterations of DNA methylation or shifts in transcriptomic profiles. The level of protein, however, has been largely neglected. We utilized mass spectrometry to investigate the proteome of CD14(+) monocytes in healthy adults reporting childhood adversity and a control group before and after psychosocial stress exposure. Particular proteins involved in (i) immune processes, such as neutrophil-related proteins, (ii) protein metabolism, or (iii) proteins related to mitochondrial biology, such as those involved in energy production processes, were upregulated in participants reporting exposure to adversity in childhood. This functional triad was further corroborated by protein interaction- and co-expression analyses, was independent of stress exposure, i.e. observed at both pre- and post-stress time points, and became evident especially in females. In line with the mitochondrial allostatic load model, our findings provide evidence for the long-term effects of childhood adversity on mitochondrial biology

C. elegans rrf-1 Mutations Maintain RNAi Efficiency in the Soma in Addition to the Germline

Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene sod-3; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools

Intestinal <i>elt-2</i> RNAi induces prominent phenotypes in <i>rrf-1</i> mutants.

<p>The indicated <i>C. elegans</i> strains were raised on bacteria expressing <i>elt-2</i> dsRNA and imaged by DIC microscopy on day 3 of adulthood. The <i>rde-1</i> strain and the <i>rde-1</i> strain expressing RDE-1 in the hypodermis (<i>lin-26p::rde-1</i>) were unaffected by RNAi, whereas all other strains tested show growth defects, a clear appearance, and an abnormal intestine. This experiment was repeated four times with ∼100 worms per strain with similar results. WT: wild-type N2 (A – Hansen lab, B – Tuck lab), <i>rrf-1(pk1417).4x: rrf-1(pk1417)</i> outcrossed 4 times to WT(A).</p

<i>rrf-1</i> mutants are resistant to RNAi against the hypodermal gene <i>bli-3</i>, but are able to process <i>gfp</i> RNAi in seam cells.

<p>The indicated <i>C. elegans</i> strains were raised on bacteria expressing <i>bli-3</i> dsRNA (A) and <i>gfp</i> dsRNA (B) and imaged by bright-field or fluorescence microscopy, respectively, on day 1 of adulthood. (A) The <i>rrf-1</i> mutants, as well as the RNAi-resistant <i>rde-1</i> strain are unaffected by <i>bli-3</i> RNAi, whereas all other strains tested show severe molting defects and varying degrees of blister formation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035428#pone.0035428.s004" target="_blank">Figure S4A</a> for additional information. This experiment was repeated three times with 100–200 worms per strain with similar results, also for several generations. WT: wild-type N2 (A – Hansen lab, B – Tuck lab), <i>rrf-1(pk1417).4x: rrf-1(pk1417)</i> outcrossed 4 times to WT(A). (B) The <i>SCMp::gfp</i> reporter is exclusively expressed in hypodermal seam cells, which are denoted with arrows. GFP expression is abolished in the wild-type background and reduced in the <i>rrf-1(pk1417)</i> background. The <i>rrf-1</i> mutant shows visibly higher GFP expression levels compared to the wild-type strain. The exposure time for the GFP channel was 100 ms. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035428#pone.0035428.s004" target="_blank">Figure S4C</a> for quantification of transgene intensity. These experiments were repeated 3 times with 30–50 worms per strain and imaging of ∼10 per experiment, with similar results.</p

RNAi efficiency in different tissues in <i>rrf-1</i> mutants.

<p>‘+’ and ‘−’ refer to the presence or absence of a phenotype in response to RNAi treatment in wild-type or <i>rrf-1</i> animals. The <i>rrf-1(pk1417), rrf-1(pk1417).4x (rrf-1(pk1417) four times outcrossed to WT(A)</i> and <i>rrf-1(ok589)</i> mutants behaved similarly to RNAi targeting endogenous genes and are combined here as <i>rrf-1</i>. For strains expressing GFP-tagged transgenes, loss of GFP expression was assayed in response to <i>gfp</i> RNAi in the <i>rrf-1(pk1417)</i> mutant only.</p><p>Phenotype abbreviations: Pro: proximal germ cell proliferation abnormal; Tum: tumorous germline; Prz: paralyzed, Unc/Twitcher: uncoordinated, sub-class, Twitcher; Gob: gut-obstructed; Clr: clear; Lva: larval arrest; Gro: slow growth; Ste: sterile; Bli: blistered; Dpy: dumpy; Bmd: body morphology defects; DTC: distal tip cell.</p>1<p>Citations refer to studies describing expression patterns and gene functions of the genes targeted by RNAi and to descriptions of the transgenes used;</p>2<p>Only the intestinal expression of <i>daf-16p::gfp::daf-16</i> was examined;</p>3<p>The intestinal, pharyngeal, and gonadal expression of <i>lgg-1p::gfp::lgg-1</i> was examined;</p>4<p>Data not shown;</p>5<p>RNAi phenotypes observed in 2^nd generation;</p>6<p>Transgene expression was only partially reduced.</p

GFP reporters in the intestine of <i>rrf-1</i> mutants are repressed by <i>gfp</i> RNAi.

<p>The indicated <i>C. elegans</i> strains were raised on bacteria expressing <i>gfp</i> dsRNA, and imaged by DIC and fluorescence microscopy (overlays shown here) on day 1 of adulthood. <i>rrf-1</i> mutants carried the <i>pk1417</i> allele. (<b>A</b>) The <i>lgg-1p::gfp::lgg-1</i> reporter is expressed in the intestine, pharynx, somatic gonad, and hypodermis. GFP expression is dramatically reduced upon treatment with <i>gfp</i> RNAi in both wild-type and <i>rrf-1</i> animals. The exposure time for the GFP channel under control conditions was 15 ms and for <i>gfp</i> RNAi 25 ms. In the <i>gfp</i> RNAi micrographs, note that the pharynx appears largely unaffected by RNAi in both wild-type and in <i>rrf-1</i> animals. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035428#pone.0035428.s005" target="_blank">Figure S5B</a>, D</b> for additional information on transgene intensity. <b>(B)</b> The <i>daf-16p::gfp::daf-16</i> reporter is expressed in the intestine, and GFP expression is abolished in the wild-type and reduced in the <i>rrf-1</i> background. The <i>rrf-1</i> mutant shows visibly higher transgene expression levels compared to the wild-type strain. The exposure time for the GFP channel was 200 ms. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035428#pone.0035428.s007" target="_blank">Figure S7A</a>, B</b> for additional information on transgene intensity. These experiments were repeated 2–3 times with 30–50 worms per strain and imaging of ∼10 per experiment, with similar results.</p

<i>rrf-1</i> mutants have increased <i>sod-3</i> mRNA levels, a normal lifespan, and normal thermo-tolerance.

<p>(<b>A</b>) The <i>sod-3 </i>mRNA levels of a mixed population of <i>C. elegans</i> wild-type (WT, N2(A)) and <i>rrf-1(pk1417).4x</i> (<i>rrf-1(pk1417)</i> mutant outcrossed four times to WT(A)) strains were determined. Bars show the mean + SEM of three independent experiments; **P<0.005 (Student’s <i>t</i>-test)<b>.</b> (<b>B</b>) Lifespan analysis of wild-type WT(A) and <i>rrf-1(pk1417).4x</i> strains at 20°C; P = 0.59, log-rank (Mantel-Cox test). This experiment has been performed three times with similar results; see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035428#pone.0035428.s007" target="_blank">Figure S7F</a></b> for additional data. (<b>C</b>) Thermo-tolerance was measured by assessing survival of WT(A) and <i>rrf-1(pk1417).4x</i> strains after 8 h incubation at 36°C; P = 0.36 (Student’s <i>t-</i>test). This experiment has been performed three times with similar results.</p