Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity

N-terminal acetylation promotes synaptonemal complex assembly in C. elegans

N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation.Wesuggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation.Wesuggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency

X-ray Fluorescence Microscopy Reveals the Role of Selenium in Spermatogenesis

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis

Deletion of <i>ALG12</i> and <i>BST1</i> induce UPR signaling and translation of UPR target genes.

<p>(<b>A</b>) Log₂ footprints and mRNA (rpkm) for a region containing <i>HAC1</i> in untreated wild-type, <i>alg12Δ</i>, <i>bst1Δ</i>, and wild-type cells treated with tunicamycin (TM). (<b>B</b>) Footprint counts (rpkm) for <i>HAC1</i> in untreated wild-type, <i>alg12Δ</i>, <i>bst1Δ</i>, and wild-type cells treated with tunicamycin (TM). Error bars indicate SEM. Measurements from biological replicates are shown. (<b>C</b>) Ribosome footprint coverage for UPR target genes. The scales of the Y axis, which shows the number of footprint reads, are independent by gene.</p

Deletion of <i>ALG12</i> and <i>BST1</i> leads to activation of the CWI signaling pathway.

<p>(<b>A</b>) Gene Ontology analysis of differentially regulated genes in <i>bst1Δ</i>. X-axis shows the number of genes in each functional category. (<b>B</b>) Sensitivity of <i>alg12Δ</i> and <i>bst1Δ</i> mutant strains to cell wall stress caused by calcofluor white (CFW) and congo red (CR). For each strain, 10× serial dilutions of logarithmically growing cells were spotted on agar plates containing indicated concentrations of the drugs. Pictures were taken after 48 h incubation at 30°C.</p

Translational control in the long-lived ER secretory pathway mutants.

<p>(<b>A</b>) Changes in protein translation in <i>alg12Δ</i>, <i>bst1Δ</i>, and wild-type cells treated with tunicamycin (TM) relative to untreated wild-type cells are shown in log₂ scale for all genes that are activated or repressed more than 1.5-fold in at least one of the strains. (<b>B</b>) Cluster analysis of log2 TE changes in <i>alg12Δ</i>, <i>bst1Δ</i>, and TM treated cells relative to untreated wild-type cells. Changes in log2 TE are shown for all genes that showed more than 1.5-fold decrease or increase in TE. (<b>C</b>) Polysome profiles of <i>alg12Δ</i>, <i>bst1Δ</i>, and TM treated cells. Long-lived deletion strains <i>alg12Δ</i> and <i>bst1Δ</i> do not show overall translational suppression.</p