Here we describe results from a proteomic study of protein-nanoparticle interactions to further the understanding of the ecotoxicological impact of silver nanoparticles (AgNPs) in the environment. We identified a number of proteins from Escherichia coli that bind specifically to bare or carbonatecoated AgNPs. Of these proteins, tryptophanase (TNase) was observed to have an especially high affinity for both surface modifications despite its low abundance in E. coli. Purified TNase loses enzymatic activity upon associating with AgNPs, suggesting that the active site may be in the vicinity of the binding site(s). TNase fragments with high affinities for both types of AgNPs were identified using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry. Differences in peptide abundance/presence in mass spectra for the two types of AgNPs suggest preferential binding of some protein fragments based on surface coating. One highbinding protein fragment contained a residue (Arg103) that is part of the active site. Ag adducts were identified for some fragments and found to be characteristic of strong binding to AgNPs rather than association of the fragments with ionic silver. These results suggest a probable mechanism for adhesion of proteins to the most commonly used commercial nanoparticles and highlight the potential effect of nanoparticle surface coating on bioavailability.

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We previously discovered that BapA, a bacterial β‐peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert β‐peptides [Geueke B, Namoto K, Seebach D & Kohler H‐PE (2005) J Bacteriol187, 5910–5917]. Here, we describe the purification and characterization of two distinct bacterial β‐peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (αβ)4 quaternary structure, the preproteins needed to be cleaved into two subunits. The two β‐peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of β‐peptides and mixed β/α‐peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a β‐amino acid at the N‐terminus; peptide substrates with an N‐terminal α‐amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N‐terminal β‐amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H‐βhXaa‐Ile‐βhTyr‐OH according to the following preferences with regard to the side chain of the N‐terminal β‐amino acid: aliphatic and aromatic > OH‐containing > hydrogen, basic and polar. Experiments with the tripeptides H‐d‐βhVal‐Ile‐βhTyr‐OH and H‐βhVal‐Ile‐βhTyr‐OH demonstrated that the two BapA enzymes preferred the peptide with the l‐configuration of the N‐terminal β‐homovaline residue as a substrate.

Thousands of man-made chemicals are constantly released into the environment by agricultural and industrial production processes, traffic, and countless other human activities. Hence, very complex mixtures of anthropogenic chemicals and the transformation products of non-persistent compounds can be found in the aquatic environment. They reflect regional input but are also influenced by long-range transport. Thus, predicting effects on organisms and assessing the quality of a specific ecosystem based on chemical analysis is a challenge. This is not only because of the wide variety of chemicals, with far ranging physicochemical properties, but also because of the sometimes very low effect levels and concerted effects caused by concentration additivity or even synergism. The situation is further complicated by the temporal variability of the exposure concentrations caused, for example, by rain events or regular daily fluctuations as seen in wastewater treatment plant effluents. The analysis of an organism's proteome allows the detection of subtle changes in the level of individual proteins in response to environmental stressors. This potentially leads to the discovery of biomarkers of exposure and helps to gain insights into underlying mechanisms of toxicity. Today, studies using environmental proteomics have investigated many organisms, ranging from microorganisms and plants to invertebrates and vertebrates. Nevertheless, proteomics is a field of environmental research still in its infancy, due to a number of caveats, such as the limited number of organisms fully covered in the sequence databases, the high genetic variability, and the dependence on environmental factors. However, it is gradually becoming an established technology. This review article highlights recent advances in the field of proteomics, mainly focusing on experimental techniques that have the potential to help us understand toxic modes of action and identify novel ecotoxicological biomarkers.

The molecular mechanisms controlling sex determination and differentiation in zebrafish (Danio rerio) are largely unknown. A genome-wide analysis may provide comprehensive insights into the processes involved. The mRNA expression in zebrafish gonads has been fairly well studied, but much less data on the corresponding protein expression are available, although the proteins are considered to be more relevant markers of gene function. Because mRNA and protein abundances rarely correlate well, mRNA profiles need to be complemented with the information on protein expression. The work presented here analyzed the proteomes of adult zebrafish gonads by a multidimensional protein identification technology, generating the to-date most populated lists of proteins expressed in mature zebrafish gonads. The acquired proteomics data partially confirmed existing transcriptomics information for several genes, including several novel transcripts. However, disagreements between mRNA and protein abundances were often observed, further stressing the necessity to assess the expression on different levels before drawing conclusions on a certain gene’s expression and function. Several gene groups expressed in a sexually dimorphic way in zebrafish gonads were identified. Their potential importance for gonad development and function is discussed. The data gained in the current study provide a basis for further work on elucidating processes occurring during zebrafish development with use of high-throughput proteomics.Electronic supplementary materialThe online version of this article (doi:10.1007/s10695-010-9464-x) contains supplementary material, which is available to authorized users.

The biosynthesis of the redox shuttle, phenazines, in Pseudomonas aeruginosa, an ubiquitous microorganism in wastewater microflora, is regulated by the 2-heptyl-3,4-dihydroxyquinoline (PQS) quorum-sensing system. However, PQS inhibits anaerobic growth of P. aeruginosa. We constructed a P. aeruginosa strain that produces higher concentrations of phenazines under anaerobic conditions by over-expressing the PqsE effector in a PQS negative ΔpqsC mutant. The engineered strain exhibited an improved electrical performance in microbial fuel cells (MFCs) and potentiostat-controlled electrochemical cells with an approximate five-fold increase of maximum current density relative to the parent strain. Electrochemical analysis showed that the current increase correlates with an over-synthesis of phenazines. These results therefore demonstrate that targeting microbial cell-to-cell communication by genetic engineering is a suitable technique to improve power output of bioelectrochemical systems.

This article is available online at http://www.jlr.org anchor has the core structure phosphatidylinositol (PI)-glucosamine (GlcN)-(Mannose) 3 -phosphoethanolamine (EtN-P), which is conserved among all species. After biosynthesis, the GPI anchor is attached posttranslationally to the newly generated C terminus of certain eukaryotic proteins destined for anchoring thereby tethering the protein to the membrane surface by the glycolipid moiety. GPI-anchored proteins can be released from the cell surface by phosphatidylinositol specifi c phospholipases and this cleavage event can induce major conformational changes on the GPIanchored protein itself ( 2 ).At least three organelles, the endoplasmic reticulum (ER), Golgi, and peroxisomes, are involved in the biosynthesis and remodeling of the GPI anchor. The biosynthesis is initiated on the outer side of the ER membrane. After the fi rst two reactions, the GPI anchor precursor is fl ipped and biosynthesis continues on the luminal side of the ER where the diacyl chains of phosphatidylinositol are then replaced by alkyl-acyl chains. This step is impaired in mutants of the peroxisomal alkyl phospholipid biosynthesis pathway ( 3 ).After protein attachment, the GPI anchor undergoes complex remodeling that begins in the ER with the removal of the inositol-linked acyl chain ( 4 ) and the remodeling of the GPI glycan part (5). Glycan remodeling is crucial for sorting GPI-anchored proteins into ER exit sites and their subsequent ER to Golgi transport ( 6 ). In mammalian cells, remodeling of the GPI anchor is then continued in the Golgi where the unsaturated fatty acid of the GPI anchor is replaced by a saturated fatty acid chain ( 7 ). Abstract Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER).After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profi le of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchordefi cient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-defi cient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by...

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