Confirmation of MDM2 substrates in mammalian cells by an <i>ex vivo</i> ubiquitination assay.

<p>HEK293T cells were transfected with the GFP-tagged substrates alone or co-transfected with Flag-tagged MDM2 (either WT or a catalytically inactive MDM2Δring mutant). The GFP-tagged substrate was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. DDX42 (A) and TP53RK (B) were ubiquitinated when MDM2 was co-expressed. TP53RK and DDX42 were ubiquitinated when MDM2 was co-expressed, whereas only very faint ubiquitination bands were observed in the MDM2Δring lane. RPL36a (C) was ubiquitinated when MDM2 was co-expressed, while weaker ubiquitination bands were observed in the MDM2Δring lane. To calculate the relative extent of RPL36a poly-ubiquitination, the poly-ubiquitination signal of RPL36a (poly-Ub-RPL36a) was quantified using ImageJ software, divided by the corresponding GFP-RPL36a signal and normalized to 1.0. HEK293T (D) cells were transfected with empty vector of GV142 expressed only GFP protein (negative control) alone or with co-transfected Flag-tagged MDM2 (either WT or a catalytically inactive MDM2Δring mutant). The GFP was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. GFP itself was not ubiquitinated by MDM2. HA-ubiquitin (E) and GFP-P53 (positive control) were co-transfected into HEK293T cells. The GFP-P53 was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. P53 was ubiquitinated when MDM2 was co-expressed. GFP tagged TP53RK (F) or RPL36a (G) were transfected into HEK293T cells alone or co-transfected with Flag-tagged MDM2. The protein content of the lysates of the transfected cells were separated and immunoblotted using the anti-GFP antibody. The expression of MDM2 induced the significant degradation of transfected TP53RK and RPL36a. When the transfected cells were treated with 10 or 20 μM MG132, the degradation of TP53RK and RPL36a were blocked. *: ubiquitination bands.</p

Screening E3 Substrates Using a Live Phage Display Library

<div><p>Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage display library as E3 substrates in <i>in vitro</i> screening. His-ubiquitinated phage, enriched with Ni-beads, could effectively infect <i>E. coli</i> for amplification. Sixteen natural potential substrates and many unnatural potential substrates of E3 MDM2 were identified through 4 independent screenings. Some substrates were identified in different independent experiments. Additionally, 10 of 12 selected candidates were ubiquitinated by MDM2 <i>in vitro</i>, and 3 novel substrates, DDX42, TP53RK and RPL36a were confirmed <i>ex vivo</i>. The whole strategy is rather simple and efficient. Non-degradation substrates can be discovered. This strategy can be extended to any E3s as long as the E3 does not ubiquitinate the empty phage.</p> </div

Schematic of the proteomic strategy using a live phage display library as E3 substrates for screening.

<p>In negative selection, the library that displayed human brain cDNA was incubated in the <i>in </i><i>vitro</i> ubiquitination system containing E1, E2, and His-tagged ubiquitin without E3. The reaction product was incubated with Ni-beads. The unbound phage was collected and amplified in <i>E</i>. <i>coli</i> BLT5403. The amplified sublibrary was used for positive selection. In positive selection, the sublibrary was incubated in the <i>in </i><i>vitro</i> ubiquitination system containing E1, E2, target E3 and His-tagged ubiquitin. The substrates that displayed on phages were mono- or poly-ubiquitinated by the E3 in the system. Then, the reaction products were incubated with Ni-beads. Phages with a His-ubiquitin tag were captured by Ni-beads. The bound phage was further eluted by imidazole and proliferated in BLT5403. The amplified sublibrary from positive selection was put into a second round of positive or negative selection. After several rounds of selection, individual clones were amplified by PCR and sequenced to obtain the potential E3-substrate coding sequences.</p

Ubiquitin ligase MDM2 does not ubiquitinate the empty phage.

<p>A. Empty phage, after the reaction as substrate in the <i>in </i><i>vitro</i> ubiquitination system containing GST-MDM2 as E3, had low nonspecific absorption to the Ni-beads (1.2×10³ PFU), as did empty phages not subjected to the reaction (1.2×10³ PFU), compared to the input (2×10⁷ PFU). B. MDM2 did not ubiquitinate the coat protein of T7 phage in Western blot analysis.</p

<i>In vitro</i> validation of candidate MDM2 substrates selected by the phage display strategy.

<p>Candidate substrates TP53RK (A), DDX42 (B), RBBP6 (C), C12orf35 (D), the unnatural substrate 1# (E), RPL15 (F), MAP2 (G), NUSCK1 (H), NOLC1 (I), RPL36a (J), PRDM2 (K), HMGN1 (L) and the negative control (M) were purified and subjected to traditional liquid ubiquitination reactions. They were then separated by SDS PAGE and immunoblotted with an anti-S tag antibody. All candidate substrates were ubiquitinated by MDM2, except PRDM2 (K) and HMGN1 (L). *: ubiquitination bands. SE: short exposure. LE: long exposure.</p

Selected baseline characteristics of 1859 participants by end-point status of knowledge in Nanjing, China.

<p>Selected baseline characteristics of 1859 participants by end-point status of knowledge in Nanjing, China.</p

The associations between change in knowledge and modification of PA-F and PA-T at study end, overall and stratified by grade and gender among urban students in Nanjing, China.

<p>The associations between change in knowledge and modification of PA-F and PA-T at study end, overall and stratified by grade and gender among urban students in Nanjing, China.</p

The relationship between awareness of knowledge and the likelihood of increase in PA-F and PA-T among urban students in Nanjing, China.

<p>The relationship between awareness of knowledge and the likelihood of increase in PA-F and PA-T among urban students in Nanjing, China.</p

Proteomic Analysis of Hepatic Tissue of <i>Cyprinus carpio</i> L. Exposed to Cyanobacterial Blooms in Lake Taihu, China

<div><p>With the rapid development of industry and agriculture and associated pollution, the cyanobacterial blooms in Lake Taihu have become a major threat to aquatic wildlife and human health. In this study, the ecotoxicological effects of cyanobacterial blooms on cage-cultured carp (<i>Cyprinus carpio</i> L.) in Meiliang Bay of Lake Taihu were investigated. Microcystins (MCs), major cyanobacterial toxins, have been detected in carp cultured at different experimental sites of Meiliang Bay. We observed that the accumulation of MCs in carp was closely associated with several environmental factors, including temperature, pH value, and density of cyanobacterial blooms. The proteomic profile of carp liver exposed to cyanobacterial blooms was analyzed using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The toxic effects of cyanobacterial blooms on carp liver were similar to changes caused by MCs. MCs were transported into liver cells and induced the excessive production of reactive oxygen species (ROS). MCs and ROS inhibited protein phosphatase and aldehyde dehydrogenase (ALDH), directly or indirectly resulting in oxidative stress and disruption of the cytoskeleton. These effects further interfered with metabolic pathways in the liver through the regulation of series of related proteins. The results of this study indicated that cyanobacterial blooms pose a major threat to aquatic wildlife in Meiliang Bay in Lake Taihu. These results provided evidence of the molecular mechanisms underlying liver damage in carp exposed to cyanobacterial blooms.</p></div

Comparison of selected baseline characteristics between those lost and followed-up.

<p>Comparison of selected baseline characteristics between those lost and followed-up.</p