Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta)

N‐glycosylation of proteins is one of the most important post‐translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca‐lization. Although the N‐linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N‐glycosylation pathway in algae. We studied the α 1,3‐glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp. (Rhodophyta) using bioinformatic and biochemical approaches. The GANAB‐gene was found to be highly conserved evolutionarily (compo‐sed of all the common features of α and β subunits) and to exhibit similar motifs consistent with that of homolog eukaryotes GANAB genes. Phylogenetic analysis revealed its wide distribution across an evolutionarily vast range of organisms; while the α subunit is highly conserved and its phylogenic tree is similar to the taxon evolutionary tree, the β subunit is less conserved and its pattern somewhat differs from the taxon tree. In addition, the activity of the red microalgal GANAB enzyme was studied, including functional and biochemical characterization using a bioassay, indicating that the enzyme is similar to other eukaryotes ortholog GANAB enzymes. A correlation between polysaccharide production and GANAB activity, indicating its involvement in polysaccharide biosynthesis, is also demonstrated. This study represents a valuable contribution toward understanding the N‐glycosylation and polysaccharide biosynthesis pathways in red microalgae.     

TDAG51 mediates the effects of insulin-like growth factor I (IGF-I) on cell survival

Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I.     

An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.     

Vanadium treatment of type 2 diabetes: A view to the future

3-Hydroxy-2-methyl-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone (maltol and ethyl maltol, respectively) have proven especially suitable as ligands for vanadyl ions, in potential insulin enhancing agents for diabetes mellitus. Both bis(maltolato)oxovanadium(IV) (BMOV), and the ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), have the desired intermediate stability for pro-drug use, and have undergone extensive pre-clinical testing for safety and efficacy. Pharmacokinetic evaluation indicates a pattern of biodistribution consistent with fairly rapid dissociation and uptake, binding to serum transferrin for systemic circulation and transport to tissues, with preferential uptake in bone. These bis-ligand oxovanadium(IV) (VOL₂) compounds have a clear advantage over inorganic vanadyl sulfate in terms of bioavailability and pharmaceutical efficacy. BEOV has now completed Phase I and has advanced to Phase II clinical trials. In the Phase I trial, a range of doses from 10 mg to 90 mg BEOV, given orally to non-diabetic volunteers, resulted in no adverse effects; all biochemical parameters remained within normal limits. In the Phase IIa trial, BEOV (AKP-020), 20 mg, daily for 28 days, per os, in seven type 2 diabetic subjects, was associated with reductions in fasting blood glucose and %HbA1c; improved responses to oral glucose tolerance testing, versus the observed worsening of diabetic symptoms in the two placebo controls.     

Myosin II Tailpiece Determines Its Paracrystal Structure, Filament Assembly Properties, and Cellular Localization

Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal non-helical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions.Non muscle myosin II (NMII)² is a major motor protein present in all cell types participating in crucial processes, including cytokinesis, surface attachment, and cell movement (1–3). NMII units are hexamers of two long heavy chains with two pairs of light chains attached. NMII heavy chain is composed of a globular head containing the actin binding and force generating ATPase domains, followed by a large coiled-coil rod that terminates with a short non-helical tailpiece (tailpiece). To carry out its cellular functions, NMII assembles into dimers and higher order filaments by interactions of the coiled-coil rod (4). The assembly process is governed by electrostatic interactions between adjacent coiled-coil rods containing alternating charged regions with specific periodicity (5–9) and is enhanced by activation of the motor domain through regulatory light chain phosphorylation (10–12). The charge periodicity also determines the register and orientation of each NMII hexamer in the filament. Additionally the C-terminal region of the coiled-coil rod contains a distinctive positively charged region and the assembly-competence domains that are crucial for proper filament assembly (5–9, 13).Three isoforms of NMII (termed NMII-A, NMII-B, and NMII-C) have been identified in mammals (14–16). Although NMII isoforms share somewhat overlapping roles, each isoform has distinctive tissue distribution and specific functions. NMII-A is important for neural growth cone retraction (17, 18) and is distributed to the front of migrating endothelial cells (19). While NMII-B participates in growth cone advancement (20) and was detected in the retracting tails of migrating endothelial cells (19). Furthermore NMII-A and NMII-B have an opposing effect on motility, since depletion of NMII-A leads to increased motility while NMII-B depletion hinders motility (21, 22). NMII-C plays a role in cytokinesis (23) and has distinct distribution in neuronal cells (24). Furthermore one NMII isoform only partly rescue cells in which siRNA was used to reduce the expression of another isoform (23, 25). This functional diversity is achieved despite a significant amino acid sequence identity between the isoforms (overall 64–80%), and the origin of these differential distributions and functions is not completely understood.Recent studies suggest that the C-terminal portion of NMII-A and NMII-B, particularly the last ∼170 amino acids, is responsible for the differential distribution of these NMII isoforms (26, 27). It was shown that swapping this region between NMII-A and NMII-B resulted in chimeric proteins, which adopted cellular localization according to the C-terminal part (26). This C-terminal ∼170 amino acid coiled-coil region contains the assembly-competence domains and other regions that are critical for filament assembly (5–9, 13) as well as the non-helical tailpiece. As the small tailpiece is also an important regulator of NMII filament assembly (27, 28) capable of changing NMII filament assembly properties; and phosphorylation of NMII tailpiece was shown to interfere with filament assembly (29–33) the tailpiece may be important for allowing NMII to perform its dynamic tasks. Because the coiled-coil regions are highly conserved between NMII isoforms, while the tailpiece is the most divergent, it is therefore a good candidate for mediating NMII isoform-specific functions. However, the exact mechanism by which the tailpiece affects NMII function is not fully understood. Here we show that the tailpiece serves as an isoform-specific control mechanism modulating filament order, assembly, and cellular function.     

Bioavailability of the isomer mixture of phytoene and phytofluene-rich alga Dunaliella bardawil in rat plasma and tissues

Dunaliella bardawil, a beta-carotene-accumulating alga was treated by the bleaching herbicide norflurazon to select sub-species rich with a mixture of 9-cis and all-trans stereoisomers of phytoene and phytofluene. The present study determines the bioavailability of phytoene and phytofluene with their stereoisomers in rats fed on a diet supplemented with Dunaliella phytoene-rich spray dried powder. Three groups of female weanling rats, eight animals each, were fed AIN diets for two weeks. The control consumed the diet as is. The experimental group was supplemented with 50 g Dunaliella powder to give phytoene/phytofluene at a level of 1 g/kg diet, and the placebo was provided with the oxidized algae free of carotenoids at the same amount. Weight gain and tissues weight of rats fed on the control diet, or on the experimental diets were statistically same. Tissue analyses were carried out by liquid chromatography at the end of two weeks feeding for vitamin A, carotenoids, phytoene and phytofluene and theirs stereoisomers. Liver analyses revealed high hepatic storage of phytoene in the experimental group. Analysis of the other tissues, adrenal, brain, heart, kidney, lung, and spleen detected small amounts of phytoene in the adrenal, kidney and spleen and in the plasma. High-pressure liquid chromatography for stereoisomeric composition was performed to all phytoene-containing tissues. The original algal diet content of 9-cis-to-all-trans ratio of 1:1 was maintained in the plasma and adrenal while in the liver, spleen and kidney the ratio was reduced to 1:3. The preferential accumulation of all-trans phytoene over 9-cis phytoene in the liver, spleen and kidney may be interpreted as indicating stronger antioxidative effect of 9-cis phytoene over the all-trans isomer or alternatively, in vivo streoisomerization of 9-cis phytoene to the all-trans structure.     

Score_set: A CAPRI benchmark for scoring protein complexes

Critical Assessment of PRedicted Interactions (CAPRI) has proven to be a catalyst for the development of docking algorithms. An essential step in docking is the scoring of predicted binding modes in order to identify stable complexes. In 2005, CAPRI introduced the scoring experiment, where upon completion of a prediction round, a larger set of models predicted by different groups and comprising both correct and incorrect binding modes, is made available to all participants for testing new scoring functions independently from docking calculations. Here we present an expanded benchmark data set for testing scoring functions, which comprises the consolidated ensemble of predicted complexes made available in the CAPRI scoring experiment since its inception. This consolidated scoring benchmark contains predicted complexes for 15 published CAPRI targets. These targets were subjected to 23 CAPRI assessments, due to existence of multiple binding modes for some targets. The benchmark contains more than 19,000 protein complexes. About 10% of the complexes represent docking predictions of acceptable quality or better, the remainder represent incorrect solutions (decoys). The benchmark set contains models predicted by 47 different predictor groups including web servers, which use different docking and scoring procedures, and is arguably as diverse as one may expect, representing the state of the art in protein docking. The data set is publicly available at the following URL: http://cb.iri.univ‐lille1.fr/Users/lensink/Score_set . Proteins 2014; 82:3163–3169. © 2014 Wiley Periodicals, Inc.     

Extracellular polysaccharide production in outdoor mass cultures of Porphyridium sp. in flat plate glass reactors

This work concerns an attempt to develop large scalecultivation of Porphyridium sp. outdoors. Theimpact on cell growth and production of solublesulphated polysaccharides of light-path length (LP)was studied in flat plate glass reactors outdoors. TheLP of the plate reactors ranged from 1.3–30 cm,corresponding to culture volumes of 3–72 L. The sidewalls of all reactors were covered, ensuring similarilluminated surfaces for all reactors. Maximal daytemperature was maintained at 26 ±1 ^°C.Growth conditions of pH (7.5), stirring (withcompressed air) and mineral nutrients, were optimal.Maximal volumetric concentration of the soluble sulfated polysaccharide (1.32 g L^-1) was obtained in winter with the smallest light-pathreactor (1.3 cm ) at a cell density of 1.37 ×10¹¹cells L^-1. Under these conditions, theviscosity of the culture medium was also highest,being inversely proportional to the culture'slight-path. Highest areal concentration of solublepolysaccharides (60 g m^-2) and areal cell density(3.01 × 10¹²m^-2) was recorded in the 20 cmLP reactor, progressively lower values being obtainedas the light path became shorter. A similar patternwas obtained for the areal productivity ofpolysaccharides, the highest being 4.15 g m^-2day^-1 (considering the total illuminated reactorsurface), produced in the 20-cm LP reactor.The main sugar composition (i.e. xylose, galactose andglucose) of the sulfated polysaccharides was similarin all reactors. As viscosity increased with timeduring culture growth, there was a substantial declinein bacterial population. Cultivation throughout mostof the year provided good evidence that a light pathlength of 20 cm in flat plate reactors under theseconditions is optimal for maximal areal solublepolysaccharide production of Porphyridium sp.     

Amino acid starvation sensitizes cancer cells to proteasome inhibition

We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines.