SIRT1 - a new mammalian substrate of nuclear autophagy

Macroautophagic/autophagic degradation of nuclear components (or nuclear autophagy) is a poorly understood area in autophagy research. We previously reported the nuclear lamina protein LMNB1 (lamin B1) as a nuclear autophagy substrate in primary human cells, stimulating the investigation of nuclear autophagy in the mammalian system. We recently reported the sirtuin protein SIRT1 as a new selective substrate of nuclear autophagy in senescence and aging. Upon senescence of primary human cells, SIRT1 degradation is mediated by a direct nuclear SIRT1-LC3 interaction, followed by nucleus-to-cytoplasm shuttling of SIRT1 and autophagosome-lysosome degradation. In vivo, SIRT1 is downregulated by lysosomes in hematopoietic and immune organs upon natural aging in mice and in aged human T cells. Our study identified another substrate of nuclear autophagy and suggests a new strategy to promote SIRT1-mediated health benefits by suppressing its autophagic degradation

Cytoplasmic chromatin triggers inflammation in senescence and cancer

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders

Characterization Of Novel Nuclear Substrates Of Mammalian Autophagy Pathway In Cellular Senescence And Aging

Autophagy is an evolutionally conserved membrane trafficking process that degrades unwanted proteins, organelles and exogenous pathogens through autophagosomes and lysosomes. In mammals, dysfunction of autophagy machinery is associated with a number of diseases and pathologies. While the majority of autophagy studies focus on its function in maintaining protein homeostasis in the cytoplasm, the role of autophagy in the nucleus is less known. In the first part of my dissertation research, I have explored the mechanism of autophagy in degrading nuclear lamina. We show that a key autophagy protein, LC3, associates with nuclear lamina protein Lamin B1 and chromatin, and mediates the translocation of Lamin B1 and associated chromatin fragments to the cytoplasm for degradation during cellular senescence. This study provides new insight into the nuclear function of mammalian autophagy pathway. With the initial understanding of the nuclear autophagy pathway, in the second part of my dissertation, I have investigated the mechanism underlying the loss of SIRT1, a critical nuclear regulator of cell metabolism and aging, in the context of cellular senescence and in vivo aging. We demonstrate that nuclear SIRT1 is degraded through autophagy machinery in senescent human fibroblasts and certain tissues of aged mice. During senescence, SIRT1 is recognized as an autophagy substrate and undergoes nucleus-to-cytoplasm transportation. The autophagy protein LC3 interacts with SIRT1 to facilitate its degradation process, while disruption of LC3-SIRT1 association rescues SIRT1 downregulation. Moreover, SIRT1 is downregulated in aged mouse spleen, testis and hematopoietic stem and progenitor cells through lysosomal degradation. Given the important roles of SIRT1 in metabolism and aging, this study sheds light on a potential strategy to maintain SIRT1 protein levels to improve SIRT1 function and promote healthy lifespan. Overall, my dissertation studies characterize two major nuclear substrates of the autophagy pathway, contribute to our knowledge in the nuclear aspect of mammalian autophagy machinery and sirtuin biology, and suggest a new perspective in pharmaceutical design of anti-aging compounds

Genetically modified soybean lines exhibit less transcriptomic variation compared to natural varieties

ABSTRACTGenetically modified (GM) soybeans provide a huge amount of food for human consumption and animal feed. However, the possibility of unexpected effects of transgenesis has increased food safety concerns. High-throughput sequencing profiling provides a potential approach to directly evaluate unintended effects caused by foreign genes. In this study, we performed transcriptomic analyses to evaluate differentially expressed genes (DEGs) in individual soybean tissues, including cotyledon (C), germ (G), hypocotyl (H), and radicle (R), instead of using the whole seed, from four GM and three non-GM soybean lines. A total of 3,351 DEGs were identified among the three non-GM soybean lines. When the GM lines were compared with their non-GM parents, 1,836 to 4,551 DEGs were identified. Furthermore, Gene Ontology (GO) analysis of the DEGs showed more abundant categories of GO items (199) among non-GM lines than between GM lines and the non-GM natural varieties (166). Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most KEGG pathways were the same for the two types of comparisons. The study successfully employed RNA sequencing to assess the differences in gene expression among four tissues of seven soybean varieties, and the results suggest that transgenes do not induce massive transcriptomic alterations in transgenic soybeans compared with those that exist among natural varieties. This work offers empirical evidence to investigate the genomic-level disparities induced by genetic modification in soybeans, specifically focusing on seed tissues

Concatameric constructs suggest an intra-subunit interaction.

<p>(A) Predicted number of intra-subunit and inter-subunit disulfide bond sites in the receptor construct. In each diagram, H and S mean His33 and Ser345, respectively. C means cysteine substitution. A circle indicates one subunit. Three subunits make up a receptor and are numbered 1, 2 and 3. In the monomer, each subunit has one N terminus and one C terminus. The concatameric constructs have only one N terminus and one C terminus. Figures (B), (C), (D), (E) and (F) present the effects of DTT and H₂O₂ on the H33C/S345C monomer, trimer CC-CC-CC, trimer HC-CS-HS, trimer CC-HS-HS, and trimer HC-CC-CS, respectively. After stable responses were evoked by 30 μM ATP (black bar), the cells were incubated in 10 mM DTT for 5 min (first arrow) and were then evoked by 30 μM ATP plus 10 mM DTT (white bar). After stable currents were obtained, cells were incubated with 0.3% H₂O₂ (second arrow) for 3 min to inverse the effects of DTT, after which the cells were evoked by 30 μM ATP plus 0.3% H₂O₂ (grey bar). The gaps indicate 3-min time intervals between ATP applications. The same protocol was applied to the H33C/S345C monomer and four different concatameric constructs. For (B), (C), (D), (E), and (F), all currents were measured at least twice to obtain stability. (G) Summary of relative current changes in (B), (C), (D), (E), and (F) after DTT application. All currents were normalised to those measured prior to application of DTT (<i>n</i>  =  3-10 cells for each case). For (G), * (<i>P</i>< 0.05), values are significantly different from that observed for trimer HC-CS-HS. ** (<i>P</i>< 0.01), values are significantly different from that observed for trimer HC-CS-HS.</p

Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

<div><p>The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC₅₀ for H33C/S345C before dithiothreitol treatment was ∼2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC₅₀ to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.</p></div

Disulfide bond formation between V48C and I328C alters channel opening.

<p>(A) Effect of DTT and H₂O₂ on V48C/I328C double mutant. The same protocol of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070629#pone-0070629-g001" target="_blank">Figure 1B</a> was applied to this double mutant. Application of DTT caused a ∼4-fold increase in receptor current. Application of 0.3% H₂O₂ reversed the effect of DTT. (B) Summary of relative current change in V48C/I328C and rP2X2R-T after DTT application. *** (<i>P</i>< 0.001), values were significantly different from those obtained for V48C, I328C and rP2X2R-T. For (B), all currents were normalised to those measured prior to application of DTT (<i>n</i>  =  3-10 cells for each case). Figure (C) and (D) show that different concentrations of ATP evoke currents in V48C/I328C and rP2X2R-T, respectively. Both were applied the same protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070629#pone-0070629-g001" target="_blank">Figure 1F</a>. (E) Concentration-response curves generated from same experiment in (C) and (D) for rP2X2R-T (•), V48C (○), I328C (▾) and V48C/I328C before (△) and after DTT application (▪). The EC₅₀ curves of single mutant and rP2X2-T after DTT treatment are not shown for the sake of clarity, because there were no significant changes. The dotted line indicates that the value of I/I_max is equal to 0.5. For (C) and (D), the gaps indicate 3-min time intervals between each ATP application.</p

Double mutant cycle analysis for His33 and Ser345.

<p>(A) Mutant cycle analysis shows free energy changes between H33C and S345C. (B) Mutant cycle analysis shows free energy changes between V48C and I328C. (C) Mutant cycle analysis shows free energy changes between H33A and S345A. (D) Mutant cycle analysis shows free energy changes between V48A and I328A. (E) Histogram showing the calculated coupling energy (ΔΔG_INT) for the indicated pairs, H33C/S345C, V48C/I328C, H33A/S345A, V48A/I328A and F44C/A337C. The dashed line indicates the experimental error (2σ), which corresponds to ± 0.14 kcal/mol. ** (<i>P</i>< 0.01), values are significantly different from those observed for negative control F44C/A337C.</p

Cysteine mutants in rP2X2 receptor.

<p>The double mutations with asterisks are from previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070629#pone.0070629-Spelta1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070629#pone.0070629-Jiang1" target="_blank">[21]</a>, which demonstrated that none of the double mutations formed disulfide bonds. N.T. means this double mutation was not tested. Data shown in the table are the mean ± S.E.M. from the cells studied, and the number of cells studied is given by <i>n</i>.</p