Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P₋₁. Although RNase A also contains a P₋₁ subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P₋₁ subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P₋₁ subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates. Deepa Sikriwal and Divya Seth contributed equally to this work.     

UNC-80 and the NCA ion channels contribute to endocytosis defects in synaptojanin mutants

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.     

COX-2 expression in cystic kidneys is dependent on dietary n-3 fatty acid composition

Dietary n-3 fatty acids generally attenuate elevated cyclooxygenase-2 (COX-2) levels in disease states. However, models of renal cystic disease (RCD) exhibit reduced renal COX-2 expression. Therefore, the in vivo regulation of COX-2 expression by dietary n-3 fatty acids was examined. In archived tissues from dietary studies, COX-2 protein and gene expression was up-regulated in diseased pcy mouse and Han:SPRD-cy rat kidneys when given diets containing eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), but not those containing -linolenic acid (ALA), compared to control diets with linoleic acid (LA). The presence of disease was necessary to elicit these effects as COX-2 expression was unaltered by diet in normal kidneys. The effects were specific for COX-2, since COX-1 levels were unaltered by these dietary manipulations in either model. Thus, in RCD, diets containing EPA and DHA but not ALA appear to specifically up-regulate renal COX-2 gene and protein levels in vivo.     

P2Y receptors in Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta‐amyloid peptides (Aβ) and neuronal death in brain regions involved in learning and memory. Although Aβ is neurotoxic, the primary mechanisms by which Aβ affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aβ have revealed that Aβ has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aβ and contributes to AD pathology. In recent years, Aβ has been shown to affect ATP release from brain and blood cells and alter the expression of G protein‐coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aβ production and elimination, neuroinflammation, neuronal function and cerebral blood flow.     

From microRNAs to targets: pathway discovery in cell fate transitions

MicroRNAs (miRNAs) are 22 nt non-coding RNAs that regulate expression of downstream targets by messenger RNA (mRNA) destabilization and translational inhibition. A large number of eukaryotic mRNAs are targeted by miRNAs, with many individual mRNAs being targeted by multiple miRNAs. Further, a single miRNA can target hundreds of mRNAs, making these small RNAs powerful regulators of cell fate decisions. Such regulation by miRNAs has been observed in the maintenance of the embryonic stem cell (ESC) cell cycle and during ESC differentiation. MiRNAs can also promote the dedifferentiation of somatic cells to induced pluripotent stem cells. During this process they target multiple downstream genes, which represent important nodes of key cellular processes. Here, we review these findings and discuss how miRNAs may be used as tools to discover novel pathways that are involved in cell fate transitions using dedifferentiation of somatic cells to induced pluripotent stem cells as a case study.     

Nonsteroidal 2,3-dihydroquinoline glucocorticoid receptor agonists with reduced PEPCK activation

Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.     

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals.     

Climate change and the risks associated with delayed breeding in a tropical wild bird population

There is growing evidence of changes in the timing of important ecological events, such as flowering in plants and reproduction in animals, in response to climate change, with implications for population decline and biodiversity loss. Recent work has shown that the timing of breeding in wild birds is changing in response to climate change partly because individuals are remarkably flexible in their timing of breeding. Despite this work, our understanding of these processes in wild populations remains very limited and biased towards species from temperate regions. Here, we report the response to changing climate in a tropical wild bird population using a long-term dataset on a formerly critically endangered island endemic, the Mauritius kestrel. We show that the frequency of spring rainfall affects the timing of breeding, with birds breeding later in wetter springs. Delays in breeding have consequences in terms of reduced reproductive success as birds get exposed to risks associated with adverse climatic conditions later on in the breeding season, which reduce nesting success. These results, combined with the fact that frequency of spring rainfall has increased by about 60 per cent in our study area since 1962, imply that climate change is exposing birds to the stochastic risks of late reproduction by causing them to start breeding relatively late in the season.