Synthesis,characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.     

Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium

Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess.     

Trends in single trait dispersion between early-mid successional stages: the importance of species pool extension and habitat scale

Aims Are there trends of increasing/decreasing dispersion of single, categorical traits related to early/late-successional species between stages of community development? If yes, are these trends dependent on species pool extension and habitat scale? Is there a consistent reduction in single trait convergence or divergence in any seral stage when scaling down from ecological to local species pool?Methods Presence of all vascular species rooted within plots of 5 × 5 m was recorded in assemblages of exposed mining spoils (EMS) and heathlands (HTL), which form a chronosequence on two abandoned ore tailing heaps located close to each other in the south-eastern Carpathians (Romania). Fifteen nominal, trait attributes of plant species co-occurring in the two seral assemblages were collected from available databases and subsequently classified as either successionally 'pioneer' or 'mature'. The strength of single trait convergence or divergence was estimated by comparison with null plant assemblages at patch type (meta-community) level by reference to the ecological or local species pool, and at community level.Important findings At patch type level, all pioneer and mature trait attributes (apart from short life span), with significant variation between the two seral stages, increased and, respectively, decreased in dispersion irrespective of species pool extension. However, these trends were more conspicuous when using the ecological species pool, very likely due to relaxation in abiotic filtering and dispersal limitation. At community level, no consistent trends were observed between EMS and HTL assemblages, probably because most trait attributes were sorted by microenvironmental filters displaying high variation, like topography or habitat patch geometry. In both seral stages, there was a general weakening of trait convergence or divergence at patch type level when scaling down from the ecological to the local species pool, which was due to niche space contraction. At community level, there was a trend of rise in dispersion of pioneer attributes along the observed chronosequence, presumably imputable to increasing competition for light and underground water, but an opposite trend of dispersion drop in mature attributes was not so evident. Based on these findings, we proposed two rules of thumb concerning the expected changes in dispersion of trait attributes at patch level along successions and between levels of species pool extension. In conclusion, trends in the successional dynamics of pioneer and mature trait dispersion are clearly detectable at meta-community level, especially by reference to the ecological species pool. Habitat scale and species pool extension are key factors to consider and report when estimating the magnitude of single trait dispersion.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

Quantitative Site-specific Phosphorylation Dynamics of Human Protein Kinases during Mitotic Progression

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.Reversible phosphorylation is a ubiquitous posttranslational protein modification that is involved in the regulation of almost all biological processes (1–3). In human, 518 protein kinases have been identified in the genome that phosphorylate the majority of cellular proteins and increase the diversity of the proteome by severalfold (4). Addition of a phosphate group to a protein can alter its structural, catalytic, and functional properties; hence, kinases require tight regulation to avoid unspecific phosphorylation, which can be deleterious to cells (5–7). As a result, cells use a variety of mechanisms to ensure proper regulation of kinase activities (8). Importantly, most kinases are also in turn regulated through autophosphorylation and phosphorylation by other kinases, thus generating complex phosphorylation networks. In particular, phosphorylation on activation segments is a common mechanism to modulate kinase activities (9–11), but additional phosphorylation sites are also frequently required for fine tuning of kinase localizations and functions (12). Some kinases contain phosphopeptide binding domains that recognize prephosphorylated sites on other kinases, resulting in processive phosphorylation and/or targeting of kinases to distinct cellular locations (13–16). Because such priming phosphorylation events depend on the activities of the priming kinases, these motifs act as conditional docking sites and restrict the interaction with docking kinases to a particular point in time and physiological state. In addition, phosphorylation sites may act through combinatorial mechanisms or through cross-talk with other posttranslational modifications (PTMs)¹ (17, 18), thus further increasing the complexity of kinase regulatory networks.Regulation of kinases is of particular interest in mitosis as most of the mitotic events are regulated by reversible protein phosphorylation (19). During mitosis, error-free segregation of sister chromatids into the two daughter cells is essential to ensure genomic stability. Physically, this process is carried out by the mitotic spindle, a highly dynamic microtubule-based structure. After entry into mitosis, the major microtubule-organizing centers in animal cells, the centrosomes, start to increase microtubule nucleation and move to opposite poles of the cell. Throughout prometaphase, microtubules emanating from centrosomes are captured by kinetochores, protein complexes assembled on centromeric chromosomal DNA. This eventually leads to the alignment of all chromosomes in a metaphase plate. Because proper bipolar attachment of chromosomes to spindle microtubules is essential for the correct segregation of chromosomes, this critical step is monitored by a signaling pathway known as the spindle assembly checkpoint (SAC) (20). This checkpoint is silenced only after all chromosomes have attached to the spindle in a bioriented fashion, resulting in the synchronous segregation of sister chromatids during anaphase. Simultaneously, a so-called central spindle is formed between the separating chromatids, and the formation of a contractile ring initiates cytokinesis. Finally, in telophase, the chromosomes decondense and reassemble into nuclei, whereas remnants of the central spindle form the midbody, marking the site of abscission. Cyclin-dependent kinase 1 (Cdk1), an evolutionarily conserved master mitotic kinase, is activated prior to mitosis and initiates most of the mitotic events. Cdk1 works in close association with other essential mitotic kinases such as Plk1, Aurora A, and Aurora B for the regulation of mitotic progression (19, 21–24). Plk1 and Aurora kinases dynamically localize to different subcellular locations to perform multiple functions during mitosis and are phosphorylated at several conserved sites. Although little is known about the precise roles of these phosphorylation sites, emerging data indicate that they are involved in regulating localization-specific functions (25, 26). Furthermore, the kinases Bub1, BubR1, and TTK (Mps1) and kinases of the Nek family play important roles in maintaining the fidelity and robustness of mitosis (19). Recently, a genome-wide RNA-mediated interference screen identified M phase phenotypes for many kinases that have not previously been implicated in cell cycle functions, indicating that additional kinases have important mitotic functions (27).Although protein phosphorylation plays a pivotal role in the regulation of cellular networks, many phosphorylation events remain undiscovered mainly because of technical limitations (28). The advent of mass spectrometry-based proteomics along with developments in phosphopeptide enrichment methods has enabled large scale global phosphoproteomics studies (29, 30). However, the number of phosphorylation sites identified on kinases is limited compared with other proteins because of their frequently low expression levels. To overcome this problem, small inhibitor-based kinase enrichment strategies were developed, resulting in the identification of more than 200 kinases from HeLa cell lysates (31, 32). This method was also used recently to compare the phosphokinomes during S phase and M phase of the cell cycle, resulting in the identification of several hundreds of M phase-specific kinase phosphorylation sites (31). In the present study, we address the dynamics of the phosphokinome during mitotic progression using large scale cell synchronization at three distinct mitotic stages, small inhibitor-based kinase enrichment, and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry. Thus, we determined the mitotic phosphorylation dynamics of more than 900 kinase phosphorylation sites and identified distinctly regulated kinase interaction networks. Our results provide a valuable resource for the dynamics of the kinome during mitotic progression and give insight into the system properties of kinase interaction networks.     

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions

Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.