Proteome of human subcutaneous adipose tissue stromal vascular fraction cells versus mature adipocytes based on DIGE.

Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and preadipocytic stromal/stem cells. To date, a majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up- or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the data set determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.

Gel-based and gel-free proteomic technologies.

Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.

Regulation of insulin action by an extract of Artemisia dracunculus L. in primary human skeletal muscle culture: a proteomics approach.

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models and enhance cellular signaling in cultured cells. To determine the mechanism of action of PMI-5011, we have measured changes in protein expression in human primary skeletal muscle culture (HSMC) from subjects with Type 2 diabetes. After obtaining skeletal muscle biopsies, HSMCs were initiated, grown to confluence, and exposed to 10 microg/mL PMI 5011 overnight. Two-dimensional difference in-gel electrophoresis was used to separate proteins, and liquid chromatography mass spectrometry was used to identify differentially regulated proteins. Additionally, real-time polymerase chain reaction (PCR) was used to confirm candidate proteins identified. These data demonstrate that a well characterized botanical extract of Artemisia dracunculus L. significantly modulates proteins involved in regulating inflammatory pathways, particularly the NFkappaB complex system.

Distinct iron binding property of two putative iron donors for the iron-sulfur cluster assembly: IscA and the bacterial frataxin ortholog CyaY under physiological and oxidative stress conditions.

Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions.