A Mechanistic View of the Role of E3 in Sumoylation

Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2 ligase Ubc9 can function without E3 enzymes, albeit with lower reaction efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the subsequent sequence of events differs: in the first E2-SUMO forms a complex with the target and E3, followed by SUMO transfer to the target. In the second, Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using dynamic correlations obtained from explicit solvent molecular dynamic simulations we illustrate the key roles played by allostery in both mechanisms. Pre-existence of conformational states explains the experimental observations that sumoylation can occur without E3, even though at a reduced rate. Furthermore, we propose a mechanism for enhancement of sumoylation by E3. Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO illustrates that the E2 enzyme is already largely pre-organized for target binding and catalysis; E3 binding shifts the equilibrium and enhances these pre-existing populations. We further observe that E3 binding regulates allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target recognition

The Mechanism of Ubiquitination in the Cullin-RING E3 Ligase Machinery: Conformational Control of Substrate Orientation

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control

Diversification and Specialization of Plant RBR Ubiquitin Ligases

Background: RBR ubiquitin ligases are components of the ubiquitin-proteasome system present in all eukaryotes. They are characterized by having the RBR (RING – IBR – RING) supradomain. In this study, the patterns of emergence of RBR genes in plants are described. Methodology/Principal Findings: Phylogenetic and structural data confirm that just four RBR subfamilies (Ariadne, ARA54, Plant I/Helicase and Plant II) exist in viridiplantae. All of them originated before the split that separated green algae from the rest of plants. Multiple genes of two of these subfamilies (Ariadne and Plant II) appeared in early plant evolution. It is deduced that the common ancestor of all plants contained at least five RBR genes and the available data suggest that this number has been increasing slowly along streptophyta evolution, although losses, especially of Helicase RBR genes, have also occurred in several lineages. Some higher plants (e. g. Arabidopsis thaliana, Oryza sativa) contain a very large number of RBR genes and many of them were recently generated by tandem duplications. Microarray data indicate that most of these new genes have low-level and sometimes specific expression patterns. On the contrary, and as occurs in animals, a small set of older genes are broadly expressed at higher levels. Conclusions/Significance: The available data suggests that the dynamics of appearance and conservation of RBR genes is quite different in plants from what has been described in animals. In animals, an abrupt emergence of many structurall

Following Ariadne's thread: a new perspective on RBR ubiquitin ligases

Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field

Remittances and Household Behavior in the Philippines

As one of the world's largest recipients of remittances, the Philippines received remittances roughly 12% of its gross domestic product in 2008. Remittances have become the single most important source of foreign exchange to the economy and a significant source of income for recipient families. Using the instrument variable estimation technique, this study examines the role of remittances in increasing household consumption and investment and thereby their potential for rebalancing economic growth and creating long-term human and capital investment. The results indicate that remittances negatively influence the share of food consumption in the total expenditure. However, unlike previous studies, the estimations show that remittances to the Philippines do not have a significant influence on other key items of consumption or investment such as spending on education and health care. A further analysis using logistical regression shows that remittances help to lift households out of poverty. Remittances thus may help in fighting poverty in the Philippines but not in rebalancing growth, especially in the long run

Proposed intervention program for deaf and hard-of-hearing high school students with behavior problems

The present investigation generally aimed to evolve a proposed school-based intervention program and home-based intervention program. The study focused on the sophomore high school students at the Philippine School for the Deaf. In order to achieve the objectives of the study, the descriptive developmental method was utilized for the research. The respondents were the teachers and parents of the second year high school students. The respondents respective views regarding the students behavior problem in school and at home were investigated using standardized inventory tests the Sutter-Eyberg Student Behavior Inventory (teacher rating scale) and the Eyberg Child Behavior Inventory (parent rating scale). The students who were identified as having behavior problems based on the instruments used were the main focus of the study. The population of the second year high school students at Philippine School for the Deaf consisted of 84 students with 8 teachers handling them. Each teacher rated the students in her class using the standardized inventory test for teachers. Of the 84 study population, 68 inventory tests were retrieved. One parent per child was given the standardized inventory test for parents and 50 were retrieved. A total of 58 respondents consisting of 50 parents and 8 teachers participated in the assessment of behavior problem among the sampled population and a total of 118 students had been rated by the respondents. The Intensity Scale had a higher percentage than that of the Problem Scale as perceived by the teachers. This suggests that the teacher may be indifferent to the vi child\u27s problems or may be defensive about being able to manage classroom behavior. The Problem Scale had a greater percentage than that of the Intensity Scale as perceived by the parents who might have had low tolerance for the normal misbehaviors of the child; might have unrealistically high expectations for children\u27s conduct or an authoritarian parenting style; might have limited understanding of child\u27s behavior and the kinds of behavior problems that are developmentally appropriate; and the parents might be attempting to cope with chaotic and difficult circumstances and got overwhelmed by stressors of child rearing. Proposed intervention programs were designed to address the most common behaviors as perceived by the respondents. Findings revealed that among the second year high school students with hearing impairment at the Philippine School for the Deaf, 42 students would need to receive school-based programs and 35 students would have to participate in home-based programs

New records of stony corals from the Philippines previously known from peripheral areas of the Indo-Pacific

New Philippine records of coral species are described here namely Cantharellus noumeae Hoeksema & Best, 1984; Stylophora kuehlmanni Scheer & Pillai, 1983; and Acropora halmaherae Wallace & Wolstenholme, 1998. Stylophora danae Milne Edwards & Haime, is also added again to the Philippine inventory to update previous lists. These species were previously reported only from areas peripheral to the Indo-West Pacific (IWP). These findings further emphasize the high diversity of coral fauna in the IWP, and in the case of Stylophora, may even suggest a shift of the genus\u27 center of diversity from the Red Sea to the IWP. © National University of Singapore

Determination of drying kinetics and sorption isotherm of black pepper (Piper Nigrum)

In the present study of food products, determination of the drying characteristics of black pepper using an oven is not yet completely established. This study aimed to determine the drying kinetics and sorption isotherm of black pepper using a convective oven at 30°C, 40°C and 50°C. The data gathered in this study were used to fit in selected mathematical models for drying kinetics and sorption isotherm. Among these models, the Midilli model (MR=0.5338exp(0.7273t-0.0551)+-0.0005t for 30°C, MR=0.5814exp(0.6293t-0.0764)+ -0.0008t for 40°C and MR=0.3187exp(1.1777t-0.0466)+ -0.0011t for 50°C) was the best fit to explain the moisture transfer in black pepper, while the GAB Model (m/0.1302=((0.1906)(0.7811)aw)/(1-(0.7811)aw)[1-(0.7811)aw+(0.1906)(0.7811)aw])) was for the equilibrium moisture content and water activity relationship. After evaluating the data, the drying characteristics of black pepper at 40°C yielded better results than 30°C and 50°C. XLSTAT and ANOVA Add-in of Microsoft Excel was the software used to compute for the necessary values in the assessment of the mathematical models for this study