Xiulan Chen

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth.

An increased population of CD4+CD25highFoxp3+ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4+ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.

Quantitative proteomics using SILAC: Principles, applications, and developments

SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight‐forward implementation, quantitative accuracy, and reproducibility over chemical labeling or label‐free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein PTMs, to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome‐wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology.

Proteomic analysis of mitochondria from Caenorhabditis elegans

Mitochondria play essential roles in cell physiological processes including energy production, metabolism, ion homeostasis, cell growth, aging and apoptosis. Proteomic strategies have been applied to the study of mitochondria since 1998; these studies have yielded decisive information about the diverse physiological functions of the organelle. As an ideal model biological system, the nematode Caenorhabditis elegans has been widely used in the study of several diseases, such as metabolic diseases and cancer. However, the mitochondrial proteome of C. elegans remains elusive. In this study, we purified mitochondria from C. elegans and performed a comprehensive proteomic analysis using the shotgun proteomic approach. A total of 1117 proteins have been identified with at least two unique peptides. Their physicochemical and functional characteristics, subcellular locations, related biological processes, and associations with human diseases, especially Parkinsons disease, are discussed. An orthology comparison was also performed between C. elegans and four other model organisms for a general depiction of the conservation of mitochondrial proteins during evolution. This study will provide new clues for understanding the role of mitochondria in the physiological and pathological processes of C. elegans.

Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture

Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal‐muscle differentiation is a multi‐step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high‐accuracy mass spectrometry (LTQ‐Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter‐or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracelluar matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up‐regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis.

Phosphoproteome analysis of rat L6 myotubes using reversed-phase C18 prefractionation and titanium dioxide enrichment.

The rat L6 myotubes is an important in vitro model system for studying signaling pathways in skeletal muscle. Exploring phosphorylation events involved in the skeletal muscle is very significant for elucidating the kinase-substrate relationship, understanding regulatory mechanisms involved in signaling pathways and providing insights into numerous cell processes. Here, we used mass spectrometry-based proteomics to conduct global phosphoproteome profiling of rat L6 myotubes. Using an efficient phosphoproteomic strategy including prefractionation of tryptic peptide mixtures with self-packed RP C18 columns, phosphopeptide enrichment with TiO(2), and 2D-LC (SCX/RP)-MS/MS analysis, a total of 2230 unique phosphopeptides from 1195 proteins were identified with a false-discovery rate of less than 1.0% using a target/decoy database searching strategy. After determining the degree of certainty of the phosphorylation site location (Ascore value >or=19), 11 Ser motifs and one Thr motif were derived from our data set using the Motif-X algorithm. Several potential signaling pathways were found in our myotubes phosphoproteome, such as the MAPK signaling pathway and the IGF-1/Insulin signaling pathway.

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl(3)-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl(3)-MeOH-0.2 M HCl (4:2:2, v/v) and CHCl(3)-MeOH-0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of (1)H-NMR, (13)C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

The profile of mitochondrial proteins and their phosphorylation signaling network in INS-1 β cells

Mitochondria have important roles in cellular physiological functions and various diseases. In pancreatic beta cells, mitochondria play a central role in glucose-stimulated insulin secretion (GSIS). To reveal the potential functions of mitochondria in the GSIS process in beta cells, shotgun proteomics was applied to profiling mitochondrial proteins and their potential phosphorylation sites in rat INS-1 cells. More than 800 proteins were assigned to mitochondria. In addition, 84 different mitochondrial phosphoproteins were identified, and 52 upstream kinases of mitochondrial phosphoproteins were predicted using bioinformatics tools. Regulation networks of mitochondrial phosphoproteins were constructed by integrating mitochondrial protein interaction networks and mitochondrial phosphorylation signaling, providing a preliminary survey of how phosphorylation signaling regulates mitochondrial function in beta cells. We present integrated resources including the protein composition and signaling pathways of mitochondria which can be used to understand the role of mitochondria in GSIS.

Mammalian mitochondrial proteomics: insights into mitochondrial functions and mitochondria-related diseases

Mitochondria are organelles that are essential for cell life and death. A huge range of pathologies, including neurodegenerative diseases, cancer, diabetes and aging, have been reported to be associated with mitochondrial dysfunction. Therefore, identification of mitochondrial proteins that are differentially expressed in these pathologies will help to further our understanding of these diseases. In recent years, great achievements have been made in mammalian mitochondrial proteomics. Here we provide an overview of the current state of knowledge with respect to the whole mitochondrial proteome, the mitochondrial subproteome, mitochondrial complexes and mitochondrial post-translational modifications. Applications of comparative mitochondrial proteomics to various pathologies that have provided clues for understanding the relationship between mitochondrial dysfunction and pathogenesis are described. We conclude that mitochondrial proteomics can be used not only to map all the components of mitochondria, but can also provide information for discovering therapeutic targets for mitochondria-related diseases.

Mitochondria in the pathogenesis of diabetes: a proteomic view.

Diabetes mellitus is a complex metabolic disorder characterized by chronic hyperglycemia due to absolute or relative lack of insulin. Though great efforts have been made to investigate the pathogenesis of diabetes, the underlying mechanism behind the development of diabetes and its complications remains unexplored. Cumulative evidence has linked mitochondrial modification to the pathogenesis of diabetes and its complications and they are also observed in various tissues affected by diabetes. Proteomics is an attractive tool for the study of diabetes since it allows researchers to compare normal and diabetic samples by identifying and quantifying the differentially expressed proteins in tissues, cells or organelles. Great progress has already been made in mitochondrial proteomics to elucidate the role of mitochondria in the pathogenesis of diabetes and its complications. Further studies on the changes of mitochondrial protein specifically post-translational modifications during the diabetic state using proteomic tools, would provide more information to better understand diabetes.

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase–substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.