Priscilla T.Y. Leung

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti-aging and metabolic effects of sirtuins.

Sirtuins are a family of NAD+‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.

Differential proteomic responses in hepatopancreas and adductor muscles of the green-lipped mussel Perna viridis to stresses induced by cadmium and hydrogen peroxide

This study aimed to reveal the proteomic responses in the hepatopancreas and adductor muscle of a common biomonitor, Perna viridis after 14-day exposure to two model chemicals, cadmium (Cd; a toxic metal) and hydrogen peroxide (H(2)O(2); a pro-oxidant), using two-dimensional gel electrophoresis coupled with multivariate statistical analyses. Unique sets of tissue-specific protein expression signatures were revealed corresponding to the two treatment groups. In the hepatopancreas, 15 and 2 spots responded to Cd and H(2)O(2) treatments respectively. 6 and 7 spots were differentially expressed in the adductor muscle for Cd and H(2)O(2) treatments, respectively. 15 differentially expressed spots were successfully identified by MALDI-TOF/TOF MS analysis. These proteins are involved in glycolysis, amino acid metabolism, energy homeostasis, oxidative stress response, redox homeostasis and protein folding, heat-shock response, and muscle contraction modulation. This is the first time, to have demonstrated that Cd exposure not only leads to substantial oxidative stress but also results in endoplasmic reticulum stress in hepatopancreas of the mussel. Such notable stress responses may be attributable to high Cd accumulation in this tissue. Our results suggested that investigations on these stress-associated protein changes could be used as a new and complementary approach in pollution monitoring by this popular biomonitor species.

Response of larval barnacle proteome to CO2-driven seawater acidification

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO(2) for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.

Proteomic response of marine invertebrate larvae to ocean acidification and hypoxia during metamorphosis and calcification.

SUMMARY Calcifying marine invertebrates with complex life cycles are particularly at risk to climate changes as they undergo an abrupt ontogenetic shift during larval metamorphosis. Although our understanding of the larval response to climate changes is rapidly advancing, the proteome plasticity involved in a compensatory response to climate change is still unknown. In this study, we investigated the proteomic response of metamorphosing larvae of the tubeworm Hydroides elegans, challenged with two climate change stressors, ocean acidification (OA; pH 7.6) and hypoxia (HYP; 2.8 mg O2 l−1), and with both combined. Using a two-dimensional gel electrophoresis (2-DE)-based approach coupled with mass spectrometry, we found that climate change stressors did not affect metamorphosis except under OA, but altered the larval proteome and phosphorylation status. Metabolism and various stress and calcification-related proteins were downregulated in response to OA. In OA and HYP combined, HYP restored the expression of the calcification-related proteins to the control levels. We speculate that mild HYP stress could compensate for the negative effects of OA. This study also discusses the potential functions of selected proteins that might play important roles in larval acclimation and adaption to climate change.

De novo transcriptomic profile in the gonadal tissues of the intertidal whelk Reishia clavigera

The intertidal whelk Reishia clavigera (formerly named as Thais clavigera) is one of the most sensitive species to organotin-associated imposex. However, the limited information on mRNA transcriptome of the species has restricted the molecular investigation on such endocrine disruption. By means of Illumina sequencing, we obtained a global de novo transcriptome from the gonadal tissues of both male and female R. clavigera, with 197,324 assembled transcripts and 151,684 condensed non-redundant transcripts. Blast hit results from the NCBIs non-redundant molluscan database showed that 28,948 transcripts were successfully annotated with significant matches at an e-value of ⩽1e(-6). Among them, 1108 transcripts were assigned a well-defined gene ontology term. As the first transcriptomic study on the gonadal tissues of R. clavigera, this study has enhanced the information of mRNA transcriptome on this species and will thus facilitate mechanistic studies of chemical contaminants (e.g., organotins) on this common biomonitor species.

Antennular morphology of the cypris larvae of the mangrove barnacle Fistulobalanus albicostatus (Cirripedia: Thoracica: Balanomorpha)

Previous studies on the ultrastructure of barnacle cypris larvae suggest that cypris morphology, especially the antennules which play a key role in selecting the final substratum for settlement, may show variations between species existing in different habitats. In the present study, the cypris morphology of the barnacle Fistulobalanus ( Balanus ) albicostatus , which predominantly lives on trunks of mangrove trees was investigated using scanning electron microscopy and compared with that of species from other habitats that have already been described. The antennular segments II and III of F. albicostatus each bear one post-axial seta. The third segment consists of an attachment disc surrounded by a thin cuticular velum and the fourth segment exhibits four sub-terminal setae and five terminal setae. The morphology of the cypris antennules of F. albicostatus is similar to other balanomorph barnacles which inhabit hard shores, revealing that the antennular morphology does not have diagnostic variations between species from the different intertidal habitats. The morphology of the caudal rami (used in the evaluation of the substratum microtopography), however, appears to vary among barnacles living on different substrata.

Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis , towards different stress stimulations

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green‐lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non‐metal oxidant (hydrogen peroxide; H2O2) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10‐I and 72 for MT10‐II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two‐dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10‐I and MT10‐II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10‐I responded promptly to Cd but had a lagged induction to H2O2 treatments, while MT10‐II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H2O2, compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.

Identification of cyclophilin as a novel allergen from Platanus orientalis pollens by mass spectrometry

Oriental plane trees are an important source of airborne allergens in cities of southwest Asia. In spite of extensive studies on Platanus acerifolia allergy, there are no reports on the molecular characterization of pollen allergens from Platanus orientalis trees. In this study, a newly recognized member of cyclophilin family with a molecular weight of 18 kDa was identified as being partly responsible for IgE reactivity of P. orientalis pollen extract.

Photosynthetic and molecular responses of the marine diatom Thalassiosira pseudonana to triphenyltin exposure.

This study aimed to investigate the responses of the marine diatom Thalassiosira pseudonana upon waterborne exposure to triphenyltin chloride (TPTCl) through determining their photosynthetic response, growth performance, and expressions of genes and proteins. Based on the growth inhibition test, the 96-h IC50 (i.e., median inhibition concentration) was found to be 1.09 μg/L (95% confidence interval (CI): 0.89-1.34 μg/L). According to photosynthetic parameters, the 96-h EC50s (i.e., median effect concentrations) were estimated at 1.54 μg/L (95% CI: 1.40-1.69 μg/L) and 1.51 μg/L (95% CI: 1.44-1.58 μg/L) for the maximum quantum yield of photosystem II (PSII) photochemistry (ΦPo) and the effective quantum yield of photochemical energy conversion in PSII (Φ2), respectively. Non-photochemical quenching in the algae was increased at low concentrations of TPTCl (0.5-1.0 μg/L) but it decreased gradually when the TPTCl concentration further increased from 1.0 to 2.5 μg/L. Results of gene expressions showed that lipid metabolism related genes were not influenced by TPTCl at 0.5 or 1.0 μg/L, while silica shell formation genes were down-regulated at 0.5 μg/L. Photosynthesis related genes were up-regulated at 0.5 μg/L TPTCl but were down-regulated at 1.0 μg/L TPTCl. Proteomics analysis revealed that relatively less proteins could be detected after exposure to 1.0 μg/L TPTCl (only about 50-60 spots) compared with that observed in the 0.5μg/L TPTCl treatment and two control groups (each with about 290-300 protein spots). At 0.5 μg/L TPTCl, five proteins were differentially expressed when compared with the seawater control and solvent control, and most of these proteins are involved in defence function to protect the biological systems from reactive oxygen species that generated by TPTCl. These proteins include oxygen-evolving enhancer protein 1 precursor, fucoxanthin chlorophyll a/c protein - LI818 clade, and mitochondrial manganese superoxide dismutase, which can function to maintain the capacity of PSII and stabilize the photosynthesis efficiency as reflected by the unchanged ΦPo and Φ2 values at 0.5 μg/L TPTCl. In contrast, the excess toxicity that caused by TPTCl at the high concentration (1.0 μg/L TPTCl) might directly damage the proteins, inhibit their expression, and/or cause the suppression of metabolism as indicated by the down-regulation of most studied proteins and genes, which could ultimately inhibit the photosynthesis and growth of the algae. Overall, this study comprehensively elucidated the toxicity effects of TPT on T. pseudonana, and partially revealed the molecular toxic mechanisms and corresponding defence responses in this model algal species.

Photosynthetic and transcriptional responses of the marine diatom Thalassiosira pseudonana to the combined effect of temperature stress and copper exposure

A 96-h exposure experiment was conducted to elucidate the toxicity responses of the marine diatom Thalassiosira pseudonana upon exposure to different temperatures and copper (Cu) concentrations. Three Cu treatments (seawater control; 200μg/L Cu, EC50 for the yield at 25°C; and 1000μg/L Cu, EC50 for growth inhibition at 25°C) were conducted against four temperatures (10°C, 15°C, 25°C and 30°C). Growth rate and photosynthetic responses showed a significant interacting thermal-chemical effect with strong synergistic responses observed at 30°C treatments. Expression of heat shock protein (hsp) was positively modulated by increasing temperatures. Hsp 90, hsp90-2 and sit1 (related to silica shell formation) were highly expressed at 30°C under 1000μg/L Cu, while the genes encoding light harvesting proteins (3HfcpA and 3HfcpB) and silaffin precursor sil3 were significantly up-regulated at 15°C under 200μg/L Cu. Our results indicated an increase Cu toxicity to T. pseudonana under high temperature and Cu dose.