Parallel synthesis and nucleic acid binding properties of C(6')-[alpha]-functionalized bicyclo-DNA

Two novel bicyclo-T nucleosides carrying a hydroxyl or a carboxymethyl substituent in C(6')-[alpha]-position were prepared and incorporated into oligodeoxynucleotides. During oligonucleotide deprotection the carboxymethyl substituent was converted into different amide substituents in a parallel way. Tm-measurements showed no dramatic differences in both, thermal affinity and mismatch discrimination, compared to unmodified oligonucleotides. The post-synthetic modification of the carboxymethyl substituent allows in principle for a parallel preparation of a library of oligonucleotides carrying diverse substituents at C(6'). In addition, functional groups can be placed into unique positions in a DNA double helix.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Alboluxin, a snake C-type lectin from Trimeresurus albolabris venom is a potent platelet agonist acting via GPIb and GPVI

Alboluxin, a potent platelet activator, was purified from Trimeresurus albolabris venom with a mass of 120 kDa non-reduced and, after reduction, subunits of 17 and 24 kDa. Alboluxin induced a tyrosine phosphorylation profile in platelets that resembles those produced by collagen and convulxin, involving the time dependent tyrosine phosphorylation of Fc receptor gamma chain (Fc gamma), phospholipase Cgamma2 (PLCgamma2), LAT and p72SYK. Antibodies against both GPIb and GPVI inhibited platelet aggregation induced by alboluxin, whereas antibodies against alpha2beta1 had no effect. Inhibition of alphaIIb beta3 reduced the aggregation response to alboluxin, as well as tyrosine phosphorylation of platelet proteins, showing that activation of alphaIIb beta3 and binding of fibrinogen are involved in alboluxin-induced platelet aggregation and it is not simply agglutination. N-terminal sequence data from the beta-subunit of alboluxin indicates that it belongs to the snake C-type lectin family. The C-type lectin subunits are larger than usual possibly due to post-translational modifications such as glycosylation. Alboluxin is a hexameric (alphabeta)3 snake C-type lectin which activates platelets via both GPIb and GPVI.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

Meprins, membrane-bound and secreted astacin metalloproteinases

The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the 'hatching' subfamily comprising alveolin, ovastacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.

Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis

OBJECTIVE: The effects of mechanical deformation of intact cartilage tissue on chondrocyte biosynthesis in situ have been well documented, but the mechanotransduction pathways that regulate such phenomena have not been elucidated completely. The goal of this study was to examine the effects of tissue deformation on the morphology of a range of intracellular organelles which play a major role in cell biosynthesis and metabolism. DESIGN: Using chemical fixation, high pressure freezing, and electron microscopy, we imaged chondrocytes within mechanically compressed cartilage explants at high magnification and quantitatively and qualitatively assessed changes in organelle volume and shape caused by graded levels of loading. RESULTS: Compression of the tissue caused a concomitant reduction in the volume of the extracellular matrix (ECM), chondrocyte, nucleus, rough endoplasmic reticulum, and mitochondria. Interestingly, however, the Golgi apparatus was able to resist loss of intraorganelle water and retain a portion of its volume relative to the remainder of the cell. These combined results suggest that a balance between intracellular mechanical and osmotic gradients govern the changes in shape and volume of the organelles as the tissue is compressed. CONCLUSIONS: Our results lead to the interpretive hypothesis that organelle volume changes appear to be driven mainly by osmotic interactions while shape changes are mediated by structural factors, such as cytoskeletal interactions that may be linked to extracellular matrix deformations. The observed volume and shape changes of the chondrocyte organelles and the differential behavior between organelles during tissue compression provide evidence for an important mechanotransduction pathway linking translational and post-translational events (e.g., elongation and sulfation of glycosaminoglycans (GAGs) in the Golgi) to cell deformation.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

Mammalian iron transporters: Families SLC11 and SLC40

This review is focused on the mammalian SLC11 and SLC40 families and their roles in iron homeostasis. The SLC11 family is composed of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the lysosomal compartment of macrophages and in the tertiary granules of neutrophils, playing a key role in innate resistance against infection by intracellular microbes. SLC11A2 is a key player in iron metabolism and is ubiquitously expressed, most notably in the proximal duodenum, immature erythroid cells, brain, placenta and kidney. Intestinal iron absorption is mediated by SLC11A2 at the apical membrane of enterocytes, followed by basolateral exit via SLC40A1. To meet the daily requirement for iron, approximately 80% of the iron comes from the breakdown of hemoglobin following macrophage phagocytosis of senescent erythrocytes (iron recycling). Both SLC11A1 and SLC11A2 play an important role in macrophage iron recycling. SLC11A2 also transports iron into the cytosol across the membrane of endocytotic vesicles of the transferrin receptor-cycle. SLC40A1 is the sole member of the SLC40 family and is involved in the only cellular iron efflux mechanism described. SLC40A1 is highly expressed in several tissues and cells that play a critical role in body iron homeostasis. The signaling pathways that regulate SLC11A2 and SLC40A1 expression at transcriptional, post-transcriptional and post-translational levels are discussed. The roles of SLC11A2 and/or SLC40A1 in iron-associated disorders such as hemochromatosis, neurodegenerative diseases, and breast cancer are also summarized.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.

A 2-D guinea pig lung proteome map

Guinea pigs represent an important model for a number of infectious and non-infectious pulmonary diseases. The guinea pig genome has recently been sequenced to full coverage, opening up new research avenues using genomics, transcriptomics and proteomics techniques in this species. In order to further annotate the guinea pig genome and to facilitate future pulmonary proteomics in this species we constructed a 2-D guinea pig proteome map including 486 protein identifications and post translational modifications (PTMs). The map has been up-loaded to the UCD 2D-PAGE open access database (http://proteomics-portal.ucd.ie/). Transit peptides, N-terminal acetylations and other PTMs are available via Peptideatlas (ftp://PASS00619:NM455hi@ftp.peptideatlas.org/). This dataset is associated with a research article published in the Journal of Proteomics [1].

Isotopic effects of nitrate photochemistry in snow: a field study at Dome C, Antarctica

Stable isotope ratios of nitrate preserved in deep ice cores are expected to provide unique and valuable information regarding paleoatmospheric processes. However, due to the post-depositional loss of nitrate in snow, this information may be erased or significantly modified by physical or photochemical processes before preservation in ice. We investigated the role of solar UV photolysis in the post-depositional modification of nitrate mass and stable isotoperatios at Dome C, Antarctica, during the austral summer of 2011/2012. Two 30 cm snow pits were filled with homogenized drifted snow from the vicinity of the base. One of these pits was covered with a plexiglass plate that transmits solar UV radiation, while the other was covered with a different plexiglass plate having a low UV transmittance. Samples were then collected from each pit at a 2–5 cm depth resolution and a 10-day frequency. At the end of the season, acomparable nitrate mass loss was observed in both pits for the top-level samples (0–7 cm) attributed to mixing with the surrounding snow. After excluding samples impacted by the mixing process, we derived an average apparent nitrogen isotopic fractionation (15" app/of role in driving the isotopic fractionation of nitrate in snow.We have estimated a purely photolytic nitrogen isotopic fractionation (15"photo) of -55.8 12.0 ‰ from the difference in the derived apparent isotopic ractionations of the two experimental fields, as both pits were exposed to similar physical processes except exposure to solar UV. This value is in close agreement with the 15" photo value of -47.9 6.8 ‰ derived in a laboratory experiment simulated for Dome C conditions (Berhanu et al., 2014). We have also observed an insensitivity of 15" with depth in the snowpack under the given experimental setup. This is due to the uniform attenuation of incoming solar UV by snow, as 15" is strongly dependent on the spectral distribution of the incoming light flux. Together with earlier work, the results presented here represent a strong body of evidence that solar UV photolysis is the most relevant post-depositional process modifying the stable isotope ratios of snow nitrate at low-accumulation sites, where many deep ice cores are drilled. Nevertheless, modeling the loss of nitrate in snow is still required before a robust interpretation of ice core records can be provided.

Cultivation strategies to enhance productivity of Pichia pastoris: A review.

Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.