Jeanette Henningsen

System-Wide Temporal Characterization of the Proteome and Phosphoproteome of Human Embryonic Stem Cell Differentiation

Dynamic phosphorylation during stem cell differentiation may control recruitment of DNA methyltransferases to silence genes that maintain pluripotency. Dynamics of the Stem Cell Phosphoproteome Understanding the signaling events that control stem cell pluripotency and self-renewal and those governing differentiation should improve our ability to develop stem cell–based therapies. Rigbolt et al. performed global quantitative proteomic and phosphoproteomic analysis of human embryonic stem cells at five time points over 24 hours of nondirected (lineage-independent) differentiation initiated by two different paradigms. They identified a common core phosphoproteome associated with both differentiation protocols, discovered several temporal patterns of phosphorylation, and made predictions about changes in the activities of kinases during the differentiation period. DNA methyltransferases (DNMTs) exhibited dynamic changes in phosphorylation status that may influence their interaction with a promoter-bound protein complex, suggesting that the phosphorylation state of DNMTs may govern their recruitment to and thus silencing of target genes, such as those that promote pluripotency, during differentiation. To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation

During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics of protein secretion. We identified and quantitatively analyzed 635 secreted proteins, including 35 growth factors, 40 cytokines, and 36 metallopeptidases. The extensive presence of these proteins that can act as potent signaling mediators to other cells and tissues strongly highlights the important role of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188 of these secreted proteins to be significantly regulated during the process of myogenesis. Comparative analyses of selected secreted proteins revealed little correlation between their mRNA and protein levels, indicating pronounced regulation by posttranscriptional mechanisms. Furthermore, analyses of the intracellular levels of members of the semaphorin family and their corresponding secretion dynamics demonstrated that the release of secreted proteins is tightly regulated by the secretory pathway, the stability of the protein, and/or the processing of secreted proteins. Finally, we provide 299 unique hydroxyproline sites mapping to 48 distinct secreted proteins and have discovered a novel hydroxyproline motif.

The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.

Background  Alterations in specific signal transduction pathways may explain the hyperproliferation and abnormal differentiation of the keratinocytes as well as the increased expression of inflammatory cytokines seen in psoriasis. Major signalling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses impinge on the mitogen‐activated protein kinases (MAPKs).

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase ζ (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

StUbEx: Stable tagged ubiquitin exchange system for the global investigation of cellular ubiquitination.

Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein-protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level. Here, we present a stable tagged ubiquitin exchange (StUbEx) cellular system in which endogenous ubiquitin is replaced with an epitope-tagged version, thereby allowing specific and efficient affinity purification of ubiquitinated proteins for global analyses of protein ubiquitination. Importantly, the overall level of ubiquitin in the cell remains virtually unchanged, thus avoiding ubiquitination artifacts associated with overexpression. The efficiency and reproducibility of the method were assessed through unbiased analysis of epidermal growth factor (EGF) signaling by quantitative mass spectrometry, covering over 3400 potential ubiquitinated proteins. The StUbEx system is applicable to virtually any cell line and can be readily adapted to any of the ubiquitin-like post-translational modifications.

Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts.

Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.

Analysis of Secreted Proteins Using SILAC

Secreted proteins serve a crucial role in the communication between cells, tissues, and organs. Proteins released to the extracellular environment exert their function either locally or at distant points of the organism. Proteins are secreted in a highly dynamic fashion by cells and tissues in the body responding to the stimuli and requirements presented by the extracellular milieu. Characterization of secretomes derived from various cell types has been performed using different quantitative mass spectrometry-based proteomics strategies, several of them taking advantage of labeling with stable isotopes. Here, we describe the use of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for the quantitative analysis of the skeletal muscle secretome during myogenesis.

Quantitative Proteomics for Investigation of Secreted Factors: Focus on Muscle Secretome

The response of cells to even slight changes in the cellular microenvironment determines the reaction of the whole organism and its ability to adapt to macroenvironmental alterations. Generally, it is well recognized that the communication between cells, tissues, and organs is critical for the maintenance of the entire body homeostasis. The different cell types that build the various organs and tissues have an enormous potential to produce proteins that once secreted in the extracellular space exert their action in an auto-, paraand/or endocrine manner. It is estimated that out of the total 20.500 protein-coding genes in human, approximately 10% encode secreted proteins (Clamp et al., 2007; Skalnikova et al., 2011). The separate and combinatorial action of these ~2200 secreted proteins can influence the biology not only at adjacent sites but also have a clear effect on the whole organism (Lin et al., 2008). The secreted factors, which can range from large proteins to short peptides, are divided into different groups or classes according to their structural properties and function. The prototypical secreted proteins are represented by the group of proteins found in the blood stream and other body fluids, the components of the extracellular matrix (ECM) and enzymes released in the intestine and stomach. An intriguing group of secreted factors comprise cell surface receptor ligands, such as hormones, growth factors, and cytokines. These proteins can exert their actions either on a limited number of responsive tissues or can act on virtually all cell types dependent on the expression of their specific receptors. It is essential to decipher in depth the signaling events that are triggered by the various hormones and growth factors to understand the general mechanisms of the biological processes that occur in a strictly controlled fashion in both space and time. The processes that secreted factors influence and directly regulate range from cellular differentiation, growth and survival to apoptosis, autophagy, and ageing. In addition, a growth factor can often exert a divergent and even opposite effect depending on the cell type and cellular state. Taken in consideration the role of secreted factors in directing biological processes, malfunction of the signaling cascades orchestrated by secreted factors can have severe consequences and lead to development of a series of complicated diseases and disorders (Flier, 2001; Pedersen, 2009; Walsh, 2009). Therefore, a comprehensive characterization of secreted molecules by different cellular subtypes, tissues, and organs can contribute to the elucidation of the physiological state of a given organism and to the determination of the