Pan-transcriptomic analysis identifies coordinated and orthologous functional modules in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum.

Diatoms are important primary producers in the ocean that thrive in diverse and dynamic environments. Their survival and success over changing conditions depend on the complex coordination of gene regulatory processes. Here we present an integrated analysis of all publicly available microarray data for the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. This resource includes shared expression patterns, gene functions, and cis-regulatory DNA sequence motifs in each species that are statistically coordinated over many experiments. These data illustrate the coordination of transcriptional responses in diatoms over changing environmental conditions. Responses to silicic acid depletion segregate into multiple distinctly regulated groups of genes, regulation by heat shock transcription factors (HSFs) is implicated in the response to nitrate stress, and distinctly coordinated carbon concentrating, CO2 and pH-related responses are apparent. Fundamental features of diatom physiology are similarly coordinated between two distantly related diatom species, including the regulation of photosynthesis, cellular growth functions and lipid metabolism. These integrated data and analyses can be explored publicly (http://networks.systemsbiology.net/diatom-portal/).

State of the human proteome in 2013 as viewed through PeptideAtlas: comparing the kidney, urine, and plasma proteomes for the biology- and disease-driven Human Proteome Project.

The kidney, urine, and plasma proteomes are intimately related: proteins and metabolic waste products are filtered from the plasma by the kidney and excreted via the urine, while kidney proteins may be secreted into the circulation or released into the urine. Shotgun proteomics data sets derived from human kidney, urine, and plasma samples were collated and processed using a uniform software pipeline, and relative protein abundances were estimated by spectral counting. The resulting PeptideAtlas builds yielded 4005, 2491, and 3553 nonredundant proteins at 1% FDR for the kidney, urine, and plasma proteomes, respectively - for kidney and plasma, the largest high-confidence protein sets to date. The same pipeline applied to all available human data yielded a 2013 Human PeptideAtlas build containing 12,644 nonredundant proteins and at least one peptide for each of ∼14,000 Swiss-Prot entries, an increase over 2012 of ∼7.5% of the predicted human proteome. We demonstrate that abundances are correlated between plasma and urine, examine the most abundant urine proteins not derived from either plasma or kidney, and consider the biomarker potential of proteins associated with renal decline. This analysis forms part of the Biology and Disease-driven Human Proteome Project (B/D-HPP) and is a contribution to the Chromosome-centric Human Proteome Project (C-HPP) special issue.

Estrogen and resveratrol regulate Rac and Cdc42 signaling to the actin cytoskeleton of metastatic breast cancer cells.

Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 microM), an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 microM, resveratrol at 5 microM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 microM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 microM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominant-negative Rac retain filopodia response to 50 microM resveratrol. Lamellipodia response to 5 microM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 microM) signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

Altering substrate specificity of phosphatidylcholine-preferring phospholipase C of Bacillus cereus by random mutagenesis of the headgroup binding site.

PLC(Bc) is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc) fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc) mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc) mutants. On the basis of available data, an aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr, or Ser greatly reduces activity toward C6PC. Thus, the substrate specificity of PLC(Bc) can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.