The application of proteomics for studying the neurorescue activity of the polyphenol (-)-epigallocatechin-3-gallate

Accumulating evidence suggests that oxidative stress resulting in reactive oxygen species generation plays a pivotal role in neurodegenerative diseases, supporting the realization of the use of radical scavengers, metal chelator agents, such as the natural polyphenols for therapy. In this study, we have focused on specific identification of proteins involved in the neurorescue activity of the green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG) in a progressive model of neuronal death, induced by long-term serum deprivation of human neuroblastoma SH-SY5Y cells. The study was designed in attempt to define biomarkers for the mechanism of action of EGCG, associated with its iron chelating properties and its ability to regulate metabolic energy balance and affect cell morphology. By using mass spectrometry analysis combined with gene expression technique, we have succeeded to identify such genes and proteins (e.g. ATP synthase mitochondrial F1 complex beta, protein kinase C epsilon, nerve vascular growth factor inducible precursor and hypoxia inducible factor-1 alpha). These results strengthen the notion that the diverse molecular signaling pathways participating in the neurorescue activity of EGCG render this multifunctional compound as potential agent to reduce risk of various neurodegenerative diseases.     

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.     

The crystal structure of the C-terminal DAP5/p97 domain sheds light on the molecular basis for its processing by caspase cleavage

DAP5/p97 (death-associated protein 5) is a member of the eukaryotic translation initiation factor 4G family. It functions as a scaffold protein promoting cap-independent translation of proteins. During apoptosis, DAP5/p97 is cleaved by caspases at position 792, yielding an 86-kDa C-terminal truncated isoform (DAP5/p86) that promotes translation of several mRNAs mediated by an internal ribosome entry site. In this study, we report the crystal structure of the C-terminal region of DAP5/p97 extending between amino acids 730 and 897. This structure consists of four HEAT-Repeats and is homologous to the C-terminal domain of eIF4GI, eIF5, and eIF2Bε. Unlike the other proteins, DAP5/p97 lacks electron density in the loop connecting α3 and α4, which harbors the caspase cleavage site. Moreover, we observe fewer interactions between these two helices. Thus, previous mapping of this site by mutation analysis is confirmed here by the resolved structure of the DAP5/p97 C-terminus. In addition, we identified the position of two conserved aromatic and acidic boxes in the structure of the DAP5/p97 C-terminus. The acidic residues in the two aromatic and acidic boxes form a continuous negatively charged patch, which is suggested to make specific interactions with other proteins such as eIF2β. The caspase cleavage of DAP5/p97 removes the subdomain carrying acidic residues in the AA-box motif, which may result in exposure of a hydrophobic surface. These intriguing structural differences between the two DAP5 isoforms suggest that they have different interaction partners and, subsequently, different functions.     

An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses

Eukaryotes contain a ubiquitous family of autophagy-associated Atg8 proteins. In animal cells, these proteins have multiple functions associated with growth, cancer, and degenerative diseases, but their functions in plants are still largely unknown. To search for novel functions of Atg8 in plants, the present report tested the effect of expression of a recombinant AtAtg8 protein, fused at its N-terminus to green fluorescent protein (GFP) and at its C-terminus to the haemagglutinin epitope tag, on the response of Arabidopsis thaliana plants to the hormones cytokinin and auxin as well as to salt and osmotic stresses. Expression of this AtAtg8 fusion protein modulates the effect of cytokinin on root architecture. Moreover, expression of this fusion protein also reduces shoot anthocyanin accumulation in response to cytokinin feeding to the roots, implying the participation of AtAtg8 in cytokinin-regulated root-shoot communication. External application of cytokinin leads to the formation of novel GFP-AtAtg8-containing structures in cells located in the vicinity of the root vascular system, which are clearly distinct in size and dynamic movement from the GFP-AtAtg8-containing autophagosome-resembling structures that were observed in root epidermis cells. Expression of the AtAtg8 fusion construct also renders the plants more sensitive to a mild salt stress and to a lesser extent to a mild osmotic stress. This sensitivity is also associated with various changes in the root architecture, which are morphologically distinct from those observed in response to cytokinin. The results imply multiple functions for AtAtg8 in different root tissues that may also be regulated by different mechanisms.     

A computational screen for C/D box snoRNAs in the human genomic region associated with Prader-Willi and Angelman syndromes

Small nucleolar RNAs (snoRNAs) play a significant role in Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), which are genomic disorders resulting from deletions in the human chromosomal region 15q11–q13. To identify snoRNAs in the region, our computational study employs key motif features of C/D box snoRNAs and introduces a complementary RNA–RNA hybridization test. We identify three previously unknown methylation guide snoRNAs targeting ribosomal 18S and 28S RNAs, and two snoRNAs targeting serotonin receptor 2C mRNA. We show that the three snoRNA candidates likely possess methylation strands complementary to, and form stable complexes with, human ribosomal RNAs. Our screen also identifies 8 other snoRNA candidates that do not pass the rRNA-complementarity and/or hybridization tests. Two of these candidates have extensive sequence similarity to HBII-52, a snoRNA that regulates the alternative splicing of serotonin receptor 2C mRNA. Six out of our eleven candidate snoRNAs are also predicted by other existing methods.     

Marine molluscs in environmental monitoring

The study reported here is part of an ongoing effort to establish sensitive and reliable biomonitoring markers for probing the coastal marine environment. Here, we report comparative measurements of a range of histological, cellular and sub-cellular parameters in molluscs sampled in polluted and reference sites along the Mediterranean coast of Israel and in the northern tip of the Gulf of Aqaba, Red Sea. Available species enabled an examination of conditions in two environmental 'compartments': benthic (Donax trunculus) and intertidal (Brachidontes pharaonis, Patella caerulea) in the Mediterranean; pelagic (Pteria aegyptia) and intertidal (Cellana rota) in the Red Sea. The methodology used provides rapid results by combining specialized fluorescent probes and contact microscopy, by which all parameters are measured in unprocessed animal tissue. The research focused on three interconnected levels. First, antixenobiotic defence mechanisms aimed at keeping hazardous agents outside the cell. Paracellular permeability was 70–100% higher in polluted sites, and membrane pumps (MXRtr and SATOA) activity was up to 65% higher in polluted compared to reference sites. Second, intracellular defence mechanisms that act to minimize potential damage by agents having penetrated the first line of defence. Metallothionein expression and EROD activity were 160–520% higher in polluted sites, and lysosomal functional activity (as measured by neutral red accumulation) was 25–50% lower. Third, damage caused by agents not sufficiently eliminated by the above mechanisms (e.g. single-stranded DNA breaks, chromosome damage and other pathological alterations). At this level, the most striking differences were observed in the rate of micronuclei formation and DNA breaks (up to 150% and 400% higher in polluted sites, respectively). The different mollusc species used feature very similar trends between polluted and reference sites in all measured parameters. Concentrating on relatively basic levels of biological organization—the molecular and cellular level—the parameters measured may have the capacity not only for biomonitoring environmental quality, but also for early warning.Communicated by H. von Westernhagen, A. Diamant     

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis

BAT‐controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet‐induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold‐stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1‐cre (BAT‐Mfn2‐KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold‐stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender‐specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP‐synthesizing fat oxidation, whereas in BAT from males, complex I‐driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT‐Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole‐body cold‐stimulated thermogenesis.     

In vitro RNA random pools are not structurally diverse: a computational analysis

In vitro selection of functional RNAs from large random sequence pools has led to the identification of many ligand-binding and catalytic RNAs. However, the structural diversity in random pools is not well understood. Such an understanding is a prerequisite for designing sequence pools to increase the probability of finding complex functional RNA by in vitro selection techniques. Toward this goal, we have generated by computer five random pools of RNA sequences of length up to 100 nt to mimic experiments and characterized the distribution of associated secondary structural motifs using sets of possible RNA tree structures derived from graph theory techniques. Our results show that such random pools heavily favor simple topological structures: For example, linear stem-loop and low-branching motifs are favored rather than complex structures with high-order junctions, as confirmed by known aptamers. Moreover, we quantify the rise of structural complexity with sequence length and report the dominant class of tree motifs (characterized by vertex number) for each pool. These analyses show not only that random pools do not lead to a uniform distribution of possible RNA secondary topologies; they point to avenues for designing pools with specific simple and complex structures in equal abundance in the goal of broadening the range of functional RNAs discovered by in vitro selection. Specifically, the optimal RNA sequence pool length to identify a structure with x stems is 20x.