Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Elucidating the role of mitochondrial-localized hepatocyte growth factor receptor in gastric oncogenesis

Gastric cancer is one of the most common malignancies and leading cause of cancer-related death worldwide. Tremendous reports have demonstrated that a wide variety of receptor tyrosine kinases play a causal role in gastric cancers. Receptor tyrosine kinase MET, aka. hepatocyte growth factor receptor (HGFR), has been frequently found dysregulated in gastric carcinomas. MET is a single-pass receptor tyrosine kinase localizing on plasma membrane and specific for hepatocyte growth factor/scatter factor (HGF/SF)1. Activation of MET/HGF regulates many cellular processes, such as proliferation, survival, motility, angiogenesis and morphogenesis. In gastric cancer, aberrant MET/HGF signaling pathway has been found dysregulate cell proliferation, contributing to angiogenesis, oncogenesis, tumor cell invasion and metastasis, and also protection from apoptosis in cancer cells. Therapeutic strategies targeting MET/HGF thus hold promise for gastric cancer. Handful of studies on MET signaling in gastric cancer have looked into the underlying mechanisms of dysregulated MET signaling in gastric oncogenesis. Recently it was reported that several RTKs including MET can translocalize into mitochondria of cancer cells. An important role of mtMET (the mitochondrial-localized MET receptor is referred to as mtMET) in cancers is suggested. In this study, we aim to understand the function and substrate of mtMET of SNU5 gastric cancer cell line. Based on preliminary data from qualitative profiling of mitochondrial proteome, we identified different components of endocytosis pathway. Functional assays using different endocytic inhibitors coupled with immunofluorescence staining approach were applied to study the translocalization of MET into mitochondria. Abundance and phosphorylation of MET in mitochondria were severely disrupted when endocytosis was inhibited. We proposed that MET is activated and endocytosed, followed by translocation of a portion of MET into mitochondria of cancer cells. Moreover, our findings have suggested the participation of other mechanisms in the mitochondrial translocalization of MET. Co-immunoprecipitation and TMT-based high-throughput quantitative proteomic approach were also adopted to identify the potential substrates of mtMET in mitochondria. Among the proteins which were identified at high confidence level, a major fraction is involved in catalytic activity and metabolic process in mitochondria. Proximity ligation assays have revealed that mtMET has direct protein-protein interaction with two novel substrates, HMGA1 and PKM2. Taken together, this report suggested a novel functional role of mtMET in regulation of energy metabolism in mitochondria. The present study has disclosed a novel paradigm of mtMET signaling in gastric cancer cells and thus contributed to the ongoing efforts to therapeutically target aberrant RTK activities in human cancers.DOCTOR OF PHILOSOPHY (SBS

Roles of mitochondria in cancer targeting therapeutics.

Dysfunctions of mitochondria have been implicated as the important factor of a wide variety of diseases. Significantly, mitochondrion has emerged as the potential targets for cancer targeting therapeutics. In particular, recent findings show that reversible protein phosphorylation, which is a crucial but largely overlooked means of mitochondrial post translational modification, regulates the mitochondrial functions in the oncogenic signaling network. Comprehensive understanding of mitochondrial phosphoproteomics will thus help in breakthrough of cancer therapy. Nonetheless the substoichiometric level of protein phosphorylation largely hinders the global analysis of mitochondrial phosphoproteome. Here we report optimization of several phosphopeptide enrichment approaches including batchwise IMAC, TiO2, ERLIC, SCX and ERLIC chromatography for efficient identification of phosphopeptides and phosphoproteins. A novel combinatorial enrichment strategy that couples SDS-PAGE to batchwise ERLIC, IMAC and TiO2 sequentially has been developed to enrich for phosphopeptides of SNU1 and SNU5 cancer cells mitochondria. Finally we identified 403 and 356 phosphoproteins in SNU1 and SNU5 mitochondria respectively. A list of 44 important phosphoproteins is summarized. These results revealed diverse crucial phosphorylation activities involved in the regulation of mitochondrial functions in cancer cells.Bachelor of Science in Biological Science

Simultaneous enrichment of plasma soluble and extracellular vesicular glycoproteins using prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) approach

Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. Although intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinically useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation with electrostatic repulsion- hydrophilic interaction chromatography, to facilitate the recovery of both glycoproteins and extracellular vesicles from nondepleted human plasma. Following prolonged ultracentrifugation, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR ≤ 1%), including 48 glycoproteins with concentrations ranging from pg to ng/ml. The novel enrichment method we report should facilitate future human plasmabased proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle- derived biomarkers, that can be used to predict clinical outcomes in human patients

Simultaneous enrichment of plasma soluble and extracellular vesicular glycoproteins using prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) approach

Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. Although intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinically useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation with electrostatic repulsion-hydrophilic interaction chromatography, to facilitate the recovery of both glycoproteins and extracellular vesicles from nondepleted human plasma. Following prolonged ultracentrifugation, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR ≤ 1%), including 48 glycoproteins with concentrations ranging from pg to ng/ml. The novel enrichment method we report should facilitate future human plasma-based proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle-derived biomarkers, that can be used to predict clinical outcomes in human patients.NMRC (Natl Medical Research Council, S’pore)Accepted versio

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, whereas the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1–S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity.NMRC (Natl Medical Research Council, S’pore)Accepted versio

Hypoxia modulates A431 cellular pathways association to tumor radioresistance and enhanced migration revealed by comprehensive proteomic and functional studies

Tumor hypoxia induces cancer cell angiogenesis, invasiveness, treatment resistance, and contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerts a coordinated effect on different molecular pathways to enhance tumor growth and survival and lead to poor clinical outcome is not fully understood. In this study, we attempt to elucidate the global protein expression and functional changes in A431 epithelial carcinoma cells induced by hypoxia and reoxygenation using iTRAQ quantitative proteomics and biochemical functional assays. Quantitative proteomics results showed that 4316 proteins were quantified with FDR1.2 fold, and DNA repair, glycolysis, integrin, glycoprotein turnover, and STAT1 pathways were perturbed by hypoxia and reoxygenation-induced oxidative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous end-joining pathway, which plays a central role in DNA repair of irradiated cells, thereby potentially contributing to the radioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 dimer, a key molecular complex in the nonhomologous end-joining pathway, was confirmed by Western blot and liquid chromatography/tandem mass spectrometry-MRM methods. Functional studies confirmed that up-regulation of glycolysis, integrin, glycoprotein synthesis, and down-regulation of STAT1 pathways during hypoxia enhanced metastastic activity of A431 cells. Migration of A431 cells was dramatically repressed by glycolysis inhibitor (2-Deoxy-d-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hydrochloride), and STAT1α overexpression that enhanced the integrin-mediated cell adhesion. These results revealed that hypoxia induced several biological processes involved in tumor migration and radioresistance and provided potential new targets for tumor therapy.Accepted versio

Enhanced separation and characterization of deamidated peptides with RP-ERLIC-based multidimensional chromatography coupled with tandem mass spectrometry

Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which affects the proteins’ structure, function, and stability. Thus, it is important to identify and quantify the products to evaluate the effects in biological systems. It is still a challenging task to distinguish between the n-Asp and isoAsp deamidation products in a proteome-wide analysis because of their similar physicochemical properties. The quantification of the isomeric deamidated peptides is also rather difficult because of their coelution/poor separation in reverse-phase liquid chromatography (RPLC). We here propose a RP-ERLIC–MS/MS approach for separating and quantifying on a proteome-wide scale the three products related to deamidation of the same peptide. The key to the method is the use of RPLC in the first dimensional separation and ERLIC (electrostatic repulsion–hydrophilic interaction chromatography) in the second, with direct online coupling to tandem MS. The coelution of the three deamidation-related peptides in RPLC is then an asset, as they are collected in the same fraction. They are then separated and identified in the second dimension with ERLIC, which separates peptides on the basis of both pI and GRAVY values. The coelution of the three products in RPLC and their efficient separation in ERLIC were validated using synthetic peptides, and the performance of ERLIC–MS/MS was tested using peptide mixtures from two proteins. Applying this sequence to rat liver tissue, we identified 302 unique N-deamidated peptides, of which 20 were identified via all three deamidation-related products and 70 of which were identified via two of them

Lysine methylation of progesterone receptor at activation function 1 regulates both ligand-independent activity and ligand sensitivity of the receptor

Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.MOE (Min. of Education, S’pore)Published versio

Enhanced Separation and Characterization of Deamidated Peptides with RP-ERLIC-Based Multidimensional Chromatography Coupled with Tandem Mass Spectrometry

Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, <i>n</i>-aspartyl, and isoaspartyl residues, which affects the proteins’ structure, function, and stability. Thus, it is important to identify and quantify the products to evaluate the effects in biological systems. It is still a challenging task to distinguish between the <i>n</i>-Asp and isoAsp deamidation products in a proteome-wide analysis because of their similar physicochemical properties. The quantification of the isomeric deamidated peptides is also rather difficult because of their coelution/poor separation in reverse-phase liquid chromatography (RPLC). We here propose a RP-ERLIC–MS/MS approach for separating and quantifying on a proteome-wide scale the three products related to deamidation of the same peptide. The key to the method is the use of RPLC in the first dimensional separation and ERLIC (electrostatic repulsion–hydrophilic interaction chromatography) in the second, with direct online coupling to tandem MS. The coelution of the three deamidation-related peptides in RPLC is then an asset, as they are collected in the same fraction. They are then separated and identified in the second dimension with ERLIC, which separates peptides on the basis of both p<i>I</i> and GRAVY values. The coelution of the three products in RPLC and their efficient separation in ERLIC were validated using synthetic peptides, and the performance of ERLIC–MS/MS was tested using peptide mixtures from two proteins. Applying this sequence to rat liver tissue, we identified 302 unique <i>N</i>-deamidated peptides, of which 20 were identified via all three deamidation-related products and 70 of which were identified via two of them