Differential Proteomics Identification of HSP90 as Potential Serum Biomarker in Hepatocellular Carcinoma by Two-dimensional Electrophoresis and Mass Spectrometry

The aim of the current study is to identify the potential biomarkers involved in Hepatocellular carcinoma (HCC) carcinogenesis. A comparative proteomics approach was utilized to identify the differentially expressed proteins in the serum of 10 HCC patients and 10 controls. A total of 12 significantly altered proteins were identified by mass spectrometry. Of the 12 proteins identified, HSP90 was one of the most significantly altered proteins and its over-expression in the serum of 20 HCC patients was confirmed using ELISA analysis. The observations suggest that HSP90 might be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of HCC. This work demonstrates that a comprehensive strategy of proteomic identification combined with further validation should be adopted in the field of cancer biomarker discovery

Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes

Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

<div><p>Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and <b>9</b> hits were selected for validation. These compounds have been taken for the second-step <i>in vitro</i> tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.</p></div

Development of a Fluorescence Polarization Based High-Throughput Assay to Identify Casitas B-Lineage Lymphoma RING Domain Regulators

<div><p>The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.</p></div

FP assay performance and post-screen analysis summary.

<p>FP assay performance and post-screen analysis summary.</p

Summary of hit compounds.

<p>Summary of hit compounds.</p

A-B. Adipocyte volume.

<p>Mice were fed ad libitum with the high-fat diet before experiments and for another 12 weeks during the experiments. Mice were treated with vehicle or indicated peptides with a daily i.p. injection at 5 mg/kg. After the experiment, isolated adipocytes were obtained from excised epididymal fat pad.</p

Indirect energy expenditure and physical activity measurements result.

<p>Indirect energy expenditure and physical activity measurements result.</p

Sequences and inhibitory activity of Cbl RING domain inhibitors derived from UbCH7 L2.

<p>Letters in bold in the sequence refer to the structure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135916#pone.0135916.g001" target="_blank">Fig 1</a>.</p><p>Sequences and inhibitory activity of Cbl RING domain inhibitors derived from UbCH7 L2.</p

Glucose tolerance test in overnight fasted vehicle and peptides treatment groups.

<p>Mice were fed ad libitum with the high-fat diet before experiments and for another 12 weeks during the experiments. At the end of the experiment, glucose tolerance tests (2 g/kg glucose i.p.) were performed in overnight fasted mice. <b>A-B</b>. Blood glucose levels after the glucose challenge (i.p. 2 g/kg). <b>C-D</b>. Insulin secretion from pancreatic islets. <b>E-F</b>. Relative insulin secretion. Data are presented as means ± SE. ** P<0.05.</p