The antiobesity factor WDTC1 suppresses adipogenesis via the CRL4 WDTC 1 E3 ligase

Abstract WDTC1/Adp encodes an evolutionarily conserved suppressor of lipid accumulation. While reduced WDTC1 expression is associated with obesity in mice and humans, its cellular function is unknown. Here, we demonstrate that WDTC1 is a component of a DDB1‐CUL4‐ROC1 (CRL4) E3 ligase. Using 3T3‐L1 cell culture model of adipogenesis, we show that disrupting the interaction between WDTC1 and DDB1 leads to a loss of adipogenic suppression by WDTC1, increased triglyceride accumulation and adipogenic gene expression. We show that the CRL4WDTC1 complex promotes histone H2AK119 monoubiquitylation, thus suggesting a role for this complex in transcriptional repression during adipogenesis. Our results identify a biochemical role for WDTC1 and extend the functional range of the CRL4 complex to the suppression of fat accumulation

A Method to Generate and Analyze Modified Myristoylated Proteins

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems

Effects of competition and phosphorus fertilization on leaf and root traits of late-successional conifers Abies fabri and Picea brachytyla

Leaf and root systems are known to show a high degree of developmental plasticity in response to the local environment. However, few studies have investigated simultaneously the leaf and root traits as affected by competition and phosphorus (P) fertilization, especially in connection with the primary succession. We investigated morphological and physiological responses to different competition treatments (infra- vs. interspecific competition) and P regimes in seedlings of Abies fabri and Picea brachytyla, collected from the late succession stage Hailuogou glacier retreat area. A. fabri had a greater total chlorophyll content and specific leaf area (SLA), higher leaf nitrogen (N) and P concentrations, as well as a higher water use efficiency (assessed by the carbon isotope composition, delta C-13) and N absorption relative to P. brachytyla under P fertilization conditions, and its total biomass responded more strongly to P fertilization, especially under interspecific competition. P fertilization decreased the specific root length (SRL) and ectomycorrhizal infection in both species and specific root tip density in P. brachytyla but it had no effect on the average root diameter. We concluded that similar changes in root characteristics, but the superior performance of above-ground traits in A. fabri in response to P availability, especially under competition, explain the greater competitive capacity of A. fabri at final stages of succession. These findings highlight the influence of soil nutrition availability and competition on the functional traits of plants and contribute to the understanding of the role of relative modifications in leaf and root traits during succession.Peer reviewe

CRL4VprBP E3 Ligase Promotes Monoubiquitylation and Chromatin Binding of TET Dioxygenases

DNA methylation at the C-5 position of cytosine (5mC) regulates gene expression and plays pivotal roles in various biological processes. The TET dioxygenases iterative oxidation of 5mC, leading to eventual demethylation intermediate. Inactivation of TET enzymes causes multi-stage developmental defects, impaired cell reprogramming and hematopoietic malignancies. However, little is known about how TET activity is regulated. Here we show that all three TET proteins bind to VprBP and are monoubiquitylated by the VprBP-DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4VprBP) on a highly conserved lysine residue. Deletion of VprBP in oocytes abrogated paternal DNA hydroxymethylation in zygotes. VprBP-mediated monoubiquitylation promotes TET binding to chromatin. Multiple recurrent TET2-inactivating mutations derived from leukemia target either the monoubiquitylation site (K1299) or residues essential for VprBP binding. Cumulatively, our data demonstrate that CRL4VprBP is a critical regulator of TET dioxygenases during development and in tumor suppression

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes

Global analysis of the rat and human platelet proteome - the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomics technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing ‘core proteome’, and the ‘evolutionary proteome’ is actually a relatively static proteome

Effects of phosphorus availability on later stages of primary succession in Gongga Mountain glacier retreat area

Intra- and interspecific competition and modifications in environmental characteristics are the main drivers of plant community dynamics, but few studies have investigated the combined effects of competition and phosphorus (P) availability on ecological succession. Seedlings of conifers Abies fabri and Picea brachytyla were collected from the late-stage Hailuogou glacier retreat area and grown under different P regimes (control and P fertilization) to investigate the impact of intra- and interspecific competition on photosynthetic capacity, resource (water, N and P) use efficiency and growth performance in two types of native soil. In the control treatment, there were no differences in the total biomass of A. fabri between the two competition patterns under either type of soil, whereas interspecific competition decreased the total biomass of P. brachytyla grown in the soil collected from A. fabri plots. However, under P fertilization, A. fabri individuals exposed to interspecific competition showed a stronger competitive ability, as their total biomass, absolute height growth rate, net photosynthetic rate, water use efficiency (delta C-13) and leaf P content were significantly higher under interspecific competition compared to intraspecific competition. No differences in these traits were detected in P. brachytyla between the two competition patterns. The results indicated that P plays an important role in determining asymmetric competition patterns among Pinaceae species. The interactive effect of interspecific competition and P availability highlighted here could influence the community composition and dynamics of plants during late stage primary succession in a glacier retreat area.Peer reviewe

A chromatin activity-based chemoproteomic approach reveals a transcriptional repressome for gene-specific silencing

Immune cells develop endotoxin tolerance (ET) after prolonged stimulation. ET increases the level of a repression mark H3K9me2 in the transcriptional-silent chromatin specifically associated with pro-inflammatory genes. However, it is not clear what proteins are functionally involved in this process. Here we show that a novel chromatin activity based chemoproteomic (ChaC) approach can dissect the functional chromatin protein complexes that regulate ET-associated inflammation. Using UNC0638 that binds the enzymatically active H3K9-specific methyltransferase G9a/GLP, ChaC reveals that G9a is constitutively active at a G9a-dependent mega-dalton repressome in primary endotoxin-tolerant macrophages. G9a/GLP broadly impacts the ET-specific reprogramming of the histone code landscape, chromatin remodeling, and the activities of select transcription factors. We discover that the G9a-dependent epigenetic environment promotes the transcriptional repression activity of c-Myc for gene-specific co-regulation of chronic inflammation. ChaC may be also applicable to dissect other functional protein complexes in the context of phenotypic chromatin architectures

Discovery of biomarker candidates for coronary artery disease from an APOE-knock out mouse model using iTRAQ-based multiplex quantitative proteomics

Due to the lack of precise markers indicative of its occurrence and progression, coronary artery disease (CAD), the most common type of heart diseases, is currently associated with high mortality in the United States. To systemically identify novel protein biomarkers associated with CAD progression for early diagnosis and possible therapeutic intervention, we employed an iTRAQ-based quantitative proteomic approach to analyze the proteome changes in the plasma collected from a pair of wild type versus apolipoprotein E knockout (APOE −/−) mice which were fed with a high fat diet. In a multiplex manner ITRAQ serves as the quantitative ‘in-spectra’ marker for ‘cross-sample’ comparisons to determine the differentially expressed/secreted proteins caused by APOE knock-out. To obtain the most comprehensive proteomic datasets from this CAD-associated mouse model we applied both MALDI and ESI-based mass spectrometric (MS) platforms coupled with two different schemes of multidimensional liquid chromatography (2D-LC) separation. We then comparatively analyzed a series of the plasma samples collected at six and twelve weeks after the mice were fed with fat diets, where the 6-week or 12-week time point represents the early or intermediate phase of the fat-induced CAD, respectively. We then categorized those proteins showing abundance changes in accordance with APOE depletion. Several proteins such as the gamma and beta chains of fibrinogen, apolipoprotein B, apolipoprotein C-I, and thrombospondin-4 were among the previously known CAD markers identified by other methods. Our results suggested that these unbiased proteomic methods are both feasible and a practical means of discovering potential biomarkers associated with CAD progression