Adelina Rogowska-Wrzesinska

Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective

Proteins can become oxidatively modified in many different ways, either by direct oxidation of amino acid side chains and protein backbone or indirectly by conjugation with oxidation products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative modifications are thought to be relevant in physiological processes, irreversible oxidative modifications are known to contribute to cellular damage and disease. The most well-studied irreversible protein oxidation is carbonylation. In this work we first examine how protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo and in vitro with an emphasis on cellular metal ion homeostasis and metal binding. We then review proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of carbonylation sites in relation to cellular metabolism using the mitochondrion as a case story.

2D gels still have a niche in proteomics

With the rapid advance of MS-based proteomics one might think that 2D gel-based proteomics is dead. This is far from the truth. Current research has shown that there are still a number of places in the field of protein and molecular biology where 2D gels still play a leading role. The aim of this review is to highlight some of these applications. Examples from our own research as well as from other published works are used to illustrate the 2D gel driven research in the areas of: 1) de novo sequencing and protein identification from organisms with no or incomplete genome sequences available; 2) alternative detection methods for modification specific proteomics; 3) identification of protein isoforms and modified proteins. With an example of the glycoprotein TIMP-1 protein we illustrate the unique properties of 2D gels for the separation and characterisation of multiply modified proteins. We also show that careful analysis of experimental and theoretical protein mass and pI can lead to the identification of unanticipated protein variants modified by for example proteolytic cleavage. Together this shows that there is an important niche for 2D gel-based proteomics, which compliments traditional LC-MS techniques for specific protein research purposes.

Insights into the cellular response triggered by silver nanoparticles using quantitative proteomics.

The use of nanoparticles in foods, materials, and clinical treatments has increased dramatically in the past decade. Because of the possibility of human exposure to nanoparticles, there is an urgent need to investigate the molecular mechanisms underlying the cellular responses that might be triggered. Such information is necessary to assess potential health risks arising from the use of nanoparticles, and for developing new formulations of next generation nanoparticles for clinical treatments. Using mass spectrometry-based proteomic technologies and complementary techniques (e.g., Western blotting and confocal laser scanning microscopy), we present insights into the silver nanoparticle-protein interaction in the human LoVo cell line. Our data indicate that some unique cellular processes are driven by the size. The 100 nm nanoparticles exerted indirect effects via serine/threonine protein kinase (PAK), mitogen-activated protein kinase (MAPK), and phosphatase 2A pathways, and the 20 nm nanoparticles induced direct effects on cellular stress, including generation of reactive oxygen species and protein carbonylation. In addition, we report that proteins involved in SUMOylation were up-regulated after exposure to 20 nm silver nanoparticles. These results were further substantiated by the observation of silver nanoparticles entering the cells; however, data indicate that this was determined by the size of the nanoparticles, since 20 nm particles entered the cells while 100 nm particles did not.

Protein modification and replicative senescence of WI-38 human embryonic fibroblasts

Oxidized proteins as well as proteins modified by the lipid peroxidation product 4‐hydroxy‐2‐nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI‐38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis‐based proteomic approach coupled with immunodetection of HNE‐, AGE‐modified and carbonylated proteins. Thirty‐seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE‐modified proteins could be explained by the senescence‐associated decreased activity of glyoxalase‐I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age‐related build‐up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP‐stimulated Lon‐like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins.

Identification of Novel Oxidized Protein Substrates and Physiological Partners of the Mitochondrial ATP-dependent Lon-like Protease Pim1

ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Pim1, a Lon-like serine protease in Saccharomyces cerevisiae, is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Pim1, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to lose integrity of mitochondrial genome, and to be respiration-deficient. Because of the severity of phenotypes associated with the depletion of Pim1, this protease appears to be an essential component of the protein quality control machinery in mitochondria and to exert crucial functions during the biogenesis of this organelle. Nevertheless, its physiological substrates and partners are not fully characterized. Therefore, we used the combination of different proteomic techniques to assess the nature of oxidized protein substrates and physiological partners of Pim1 protease under non-repressing growth conditions. The results presented here supply evidence that Pim1-mediated proteolysis is required for elimination of oxidatively damaged proteins in mitochondria.

The Potato Tuber Mitochondrial Proteome

A high-coverage potato tuber mitochondrial proteome uncovers many new proteins and functions, especially in coenzyme and iron metabolism, and many posttranslational modifications. Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum ‘Folva’) and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more “extreme” proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior. One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non–leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.

In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.

Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi.

BackgroundLeaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.ResultsWe tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade.ConclusionsPectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.

Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress.

Friedreichs ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreichs ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2muYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe-S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2muYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2muYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.