A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis
ABSTRACT: Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states,termed commitment, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.
Project description:The lysine acetylation of proteins is a major post-translational modification that plays an important regulatory role in almost every aspect of cells, both eukaryotes and prokaryotes. Vibrio parahaemolyticus, a model marine bacterium, is a worldwide cause of bacterial seafood-borne illness. Here, we conducted the ﬁrst lysine acetylome in the bacterium through combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 1413 lysine acetylation sites in 656 proteins, which account for 13.6% of the total proteins in the cells and is the highest ratio of acetyl proteins that has so far been identified in bacteria. The bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. More specifically, proteins related to protein biosynthesis and carbon metabolism are the preferential targets of lysine acetylation. Moreover, two types of acetylation motifs, a lysine or arginine at the +4/+5 position and a tyrosine, histidine, or phenylalanine at the +1/+2 position, were revealed from the analysis of the acetylome. Additionally, the protein interaction network analysis demonstrates that a wide range of interactions are modulated by protein acetylation. This study provides a significant beginning for the in-depth exploration of the physiological role of lysine acetylation in V. parahaemolyticus.
Project description:Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast, however few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A related processes in mitotic arrested cells. We identified 626 potential PP2ACdc55 substrates involved in a broad range of mitotic processes. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases’ consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases different than Cdk1. Indeed, Pkc1 and Cla4 kinases emerged as novel nodes of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in membrane trafficking and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome. In addition, we validated two new PP2ACdc55 substrates involved in early and late anaphase pathways, Slk19 and Lte1; and we also validated Zeo1, and other potential substrates, through protein interaction experiments. Finally, we performed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction.
Project description:Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in Saccharomyces cerevisiae by coupling stable isotope labelling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, whereas auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly.
Project description:CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.
Project description:Background: Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer but there are no comprehensive proteomic studies on this entity. Materials and Methods: We employed liquid chromatography coupled with tandem mass spectrometry (MS/MS) using formalin-fixed paraffin-embedded (FFPE) cSCC material to study the tumor and normal skin tissue proteomes. Ingenuity Pathway Analysis (IPA) was used to interpret the role of altered proteins in cSCC pathophysiology. Results were validated using the Human Protein Atlas and Oncomine database in silico. Results: Of 1,310 unique proteins identified, expression of an average of 144 and 88 proteins were significantly (p<0.05) increased and decreased, respectively, in the tumor samples compared to their normal counterparts. IPA analysis revealed disruptions in proteins associated with cell proliferation, apoptosis, and migration. In silico analysis confirmed that proteins corresponding to 12 antibodies, and genes corresponding to 18 proteins were differentially expressed between the two categories, validating our proteomic measurements. Conclusion: Label-free MS-based proteomics is useful for analyzing FFPE cSCC tissues.
Project description:This study aimed to reveal if peptide profiling of individual human follicular fluid could become a new non-invasive predictive biomarker for oocyte quality, and attempted to discover candidate biomarkers for fertilization. In biomarker studies, because of normal clinical or biological variabil-ity, candidate biomarkers need to be validated across a large number of samples. To ensure that the discovered biomarkers are truly associated with oocytes quality and fertilization rather than the result of variability, three independent experiments were designed with a large population. In the present study, we investigated the peptide profile of human follicular fluid with fertilized and non-fertilized oocytes from patients undergoing in vitro fertilization using proteomic analysis with liquid chromatography-tandem mass spectrometry. We additionally determined the protein identi-ties of the discovered peptide biomarkers as a first step toward understanding the pathways in which they may function.
Project description:Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many parts of the biomineralization process and its evolutionary origin remain a mystery. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms underlie its production of remarkable shells and pearls is not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins (VWAP), chondroitin sulfotransferases and regulatory elements.Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre matrices suggests that elements of chitin- and collagen-based matrices are deeply rooted and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems.
Project description:The adhesion molecule CD99 is essential for transendothelial migration (TEM) of leukocytes. Here we demonstrate by biochemical and cellular assays that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species.