25 research outputs found
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Cell-Surface Proteomics Identifies Differences in Signaling and Adhesion Protein Expression between Naive and Primed Human Pluripotent Stem Cells.
Naive and primed human pluripotent stem cells (hPSC) provide valuable models to study cellular and molecular developmental processes. The lack of detailed information about cell-surface protein expression in these two pluripotent cell types prevents an understanding of how the cells communicate and interact with their microenvironments. Here, we used plasma membrane profiling to directly measure cell-surface protein expression in naive and primed hPSC. This unbiased approach quantified over 1,700 plasma membrane proteins, including those involved in cell adhesion, signaling, and cell interactions. Notably, multiple cytokine receptors upstream of JAK-STAT signaling were more abundant in naive hPSC. In addition, functional experiments showed that FOLR1 and SUSD2 proteins are highly expressed at the cell surface in naive hPSC but are not required to establish human naive pluripotency. This study provides a comprehensive stem cell proteomic resource that uncovers differences in signaling pathway activity and has identified new markers to define human pluripotent states
The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments
INTRODUCTION: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. RESULTS: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell--along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. SUMMARY: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance
Crystal structure of thebaine 6-O-demethylase from the morphine biosynthesis pathway
Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy) is a key enzyme in the
morphine biosynthesis pathway that belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent
dioxygenases (ODD) family. Initially, T6ODM was characterized as an enzyme catalyzing Odemethylation
of thebaine to neopinone and oripavine to morphinone, however recently the substrate range
of T6ODM was expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal
structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate
(T6ODM:SIN, PDB: 5O7Y). The arrangement of the T6ODM’s active site is typical for proteins from the
ODD family, but the enzyme is characterized by a large substrate binding cavity, whose volume can
partially explain the T6ODM promiscuity. Moreover, the size of the cavity allows for binding of multiple
molecules at once, posing a question about substrate-driven specificity of the enzyme
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PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4 + T Cells
Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4+ T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4+ T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4+ T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4+ T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells
A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma
Proteomic Comparison between Maturation Drying and Prematurely Imposed Drying of Zea mays Seeds Reveals a Potential Role of Maturation Drying in Preparing Proteins for Seed Germination, Seedling Vigor, and Pathogen Resistance
We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in Maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p < 0.05) in abundance during maturation drying in embryo and endosperm, respectively. Fewer proteins (48 and 59 in embryo and endosperm, respectively) changed in abundance during prematurely Imposed drying. A number of proteins, 33 and 38 in embryo and endosperm, respectively, changed similarly in abundance during both maturation and prematurely imposed drying. Storage proteins were abundant in this group and may contribute to the acquisition of seed germinability. However, a relatively large number of proteins changed in the. embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively
Recommended from our members
PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4 + T Cells
Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4+ T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4+ T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4+ T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4+ T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells
Insights into the Cellular Response Triggered by Silver Nanoparticles Using Quantitative Proteomics
The use of nanoparticles in foods, materials, and clinical treatments has increased dramatically in the past decade. Because of the possibility of human exposure to nanoparticles, there is an urgent need to investigate the molecular mechanisms underlying the cellular responses that might be triggered. Such information is necessary to assess potential health risks arising from the use of nanoparticles, and for developing new formulations of next generation nanoparticles for clinical treatments. Using mass spectrometry-based proteomic technologies and complementary techniques (<i>e.g.</i>, Western blotting and confocal laser scanning microscopy), we present insights into the silver nanoparticle–protein interaction in the human LoVo cell line. Our data indicate that some unique cellular processes are driven by the size. The 100 nm nanoparticles exerted indirect effects <i>via</i> serine/threonine protein kinase (PAK), mitogen-activated protein kinase (MAPK), and phosphatase 2A pathways, and the 20 nm nanoparticles induced direct effects on cellular stress, including generation of reactive oxygen species and protein carbonylation. In addition, we report that proteins involved in SUMOylation were up-regulated after exposure to 20 nm silver nanoparticles. These results were further substantiated by the observation of silver nanoparticles entering the cells; however, data indicate that this was determined by the size of the nanoparticles, since 20 nm particles entered the cells while 100 nm particles did not
Genome-wide screening identifies Polycomb repressive complex 1.3 as an essential regulator of human naïve pluripotent cell reprogramming.
Uncovering the mechanisms that establish naïve pluripotency in humans is crucial for the future applications of pluripotent stem cells including the production of human blastoids. However, the regulatory pathways that control the establishment of naïve pluripotency by reprogramming are largely unknown. Here, we use genome-wide screening to identify essential regulators as well as major impediments of human primed to naïve pluripotent stem cell reprogramming. We discover that factors essential for cell state change do not typically undergo changes at the level of gene expression but rather are repurposed with new functions. Mechanistically, we establish that the variant Polycomb complex PRC1.3 and PRDM14 jointly repress developmental and gene regulatory factors to ensure naïve cell reprogramming. In addition, small-molecule inhibitors of reprogramming impediments improve naïve cell reprogramming beyond current methods. Collectively, this work defines the principles controlling the establishment of human naïve pluripotency and also provides new insights into mechanisms that destabilize and reconfigure cell identity during cell state transitions
Proteomic Comparison between Maturation Drying and Prematurely Imposed Drying of <i>Zea mays</i> Seeds Reveals a Potential Role of Maturation Drying in Preparing Proteins for Seed Germination, Seedling Vigor, and Pathogen Resistance
We
have studied the role(s) of maturation drying in the acquisition
of germinability, seedling vigor and pathogen resistance by comparing
the proteome changes in maize embryo and endosperm during mature and
prematurely imposed drying. Prematurely imposed dried seeds at 40
days after pollination (DAP) germinated almost as well as mature seeds
(at 65 DAP), but their seedling growth was slower and they were seriously
infected by fungi. A total of 80 and 114 proteins were identified
to change at least two-fold (<i>p</i> < 0.05) in abundance
during maturation drying in embryo and endosperm, respectively. Fewer
proteins (48 and 59 in embryo and endosperm, respectively) changed
in abundance during prematurely imposed drying. A number of proteins,
33 and 38 in embryo and endosperm, respectively, changed similarly
in abundance during both maturation and prematurely imposed drying.
Storage proteins were abundant in this group and may contribute to
the acquisition of seed germinability. However, a relatively large
number of proteins changed in the embryo (47 spots) and endosperm
(76 spots) specifically during maturation drying. Among these proteins,
storage proteins in the embryo and defense proteins in the endosperm
may be particularly important for seedling vigor and resistance to
fungal infection, respectively