Project description:The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network. Glycans covalently bound to proteins were employed as "baits", and proteins that interact with surface glycoproteins were connected using chemical crosslinking. Glycans on surface glycoproteins were oxidized with galactose oxidase (GAO) and sequentially surface glycoproteins together with their interactors ("prey") were enriched through hydrazide chemistry. In combination with quantitative proteomics, over 300 proteins interacting with surface glycoproteins were identified. Many important domains related to extracellular events were found on these proteins. Based on the protein-protein interaction database, we constructed the interaction network among the identified proteins, in which the hub proteins play more important roles in the interactome. Through analysis of crosslinked peptides, specific interactors were identified for glycoproteins on the cell surface. The newly developed method can be extensively applied to study glycoprotein interactions on the cell surface, including the dynamics of the surfaceome interactions in cells with external stimuli.
Project description:Detailed knowledge of cell surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Normal H9 human embryonic stem cells and the KB3 human induced pluripotent stem cell lines were analyzed by Cell Surface Capture Technology, and in parallel transcript profiles from five independent samples (i.e., Replicas 1-5 for each) were performed to facilitate protein and transcriptomic comparisons. The study compared gene expression profiles of pluripotent stem cells with Cell Surface Capture technology generated N-glycoprotein surfaceome analyses of the same cell types.
Project description:The major surface glycoprotein (Msg) is the most abundant surface protein of Pneumocystis species. Given that Msg is present on both the cyst and trophic form of Pneumocystis, and dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from P. murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or MHCII, or increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, LPS activated dendritic cells by all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, two C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.
Project description:HRG is a 75kDa heparin-binding protein non to extert gene regultaion changes on macropahges. To assess the effect of HRG on Enodothelial Cell gene regulation Human Umbilical Vein Endothelial Cells (HUVECs) were treated with Histidine-Rich Glycoprotein (HRG) 24 hour treatment with HRG. and examined for gene regulation changes versus untreated HUVECs after 6 and
Project description:Isolated human mitochondria underwent DSSO crosslinking. Following treatment, the mitochondria were separated into soluble and insoluble fractions. Both fractions were then subjected to multidimensional fractionation, including SCX and hSAX. The fractionated peptides were subsequently analyzed using CL-MS. FDR-based error estimation facilitated the creation of a comprehensive mitochondrial interaction network, revealing novel protein-protein interactions.
Project description:Trypanosoma brucei Lister 427 bloodstream forms were cultured in HMI-11 medium. Total RNA was prepared using Qiagen RNAeasy kits for single sample RNAseq to estimate VSG mRNA abundance (and not to reconstruct the transcriptome). The cDNA libraries were prepared and sequenced at the Beijing Genomics Institute (Shenzhen, China). Polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers sheared and size selected for fragments ~200 bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2. RNAseq of the resulting libraries was used for the determination of transcript abundances. Sequencing was performed on an Illumina Hiseq 2000 (Illumina, CA) platform and 90 base paired end reads obtained. Four samples were analysed: 1. Trypanosoma brucei Lister 427 expressing VSG2 2. Trypanosoma brucei Lister 427 expressing VSG6 3. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 28 days after electroporation 4. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 44 days after electroporation.