Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide

Oxidative stress is known to play important roles in engineered nanomaterial‐induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial‐mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS‐2B and nanoscale titanium dioxide. Utilizing 2‐DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2‐DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses^? canonical pathways and Ingenuity Pathway Analyses tox lists to create protein‐interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS‐2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide‐treated BEAS‐2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein‐interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.     

Proteomic analysis of different temporal expression patterns induced by N-methyl-N'-nitro-N-nitrosoguanidine treatment

We have previously shown that N-methyl- N'-nitro- N-nitrosoguanidine (MNNG), a well-known DNA alkylating agent and carcinogen, can induce multiple cellular responses with dynamic characteristics, including such responses as nontargeted mutations (NTM) at undamaged bases in DNA, up-regulation of low fidelity DNA polymerases, clustering of epidermal growth factor receptor (EGFR) and interference with its downstream signaling pathway. A dose-related analysis also revealed that different concentrations of MNNG can trigger diverse proteome changes associated with different cytotoxic effects. To further understand the dynamic cellular responses and hazardous effects caused by environmental carcinogen, a proteomic time-course study of whole cellular proteins from human amniotic epithelial cells after MNNG treatment was performed. Analysis at three different time points (3, 12 and 24 h after exposure) revealed that the major changes were taking place around 3 and 12 h after exposure. Using MALDI-TOF MS coupled with a micro solid-phase extraction (SPE) device, 90% ( n = 70) differentially expressed proteins were identified. Functional assignment revealed that many important pathways were affected, including the protein biosynthesis pathway and Ran GTPase system. We also carried out a network analysis of these proteins and the data suggest a central role for some key regulators in different pathways.     

Activation of multiple oncogene pathways: a model for experimental carcinogenesis

Evidence from experimental animal tumor models suggests that in many instances, the identity and mechanism of activation of cellular oncogenes is a function of both carcinogen and tissue specificity. In addition, the activation of no single oncogene has yet been found to be either sufficient or necessary for tumorigenesis in any particular experimental system. A hypothesis to account for these and other molecular and biological observations of experimental tumorigenesis has been developed. The hypothesis is based on the premise that multiple tissue specific groups or pathways of oncogenes exist in each cell, and that activation of all the oncogenes in any of these alternative pathways leads to transformation. It is assumed that each oncogene (which may be a member of one or more pathways) has a spontaneous and a carcinogen specific probability of activation. The latter value will vary from carcinogen to carcinogen. By modelling the spontaneous and carcinogen specific probabilities of activation of each gene, the number and identity of genes in each pathway, and the number of pathways in a particular cell type, it is possible to calculate the relative potency of carcinogens, the percentage of tumors containing each activated oncogene, the dose-response relationship, and other parameters. Use of this hypothetical model gives results consistent with experimental observations on oncogene activation in carcinogen-induced animal tumors.     

Aging enhances the production of reactive oxygen species and bactericidal activity in peritoneal macrophages by upregulating classical activation pathways

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.     

Parallel analysis of transcript and translation profiles: identification of metastasis-related signal pathways differentially regulated by drug and genetic modifications

Tumor metastasis is a complex multistep process normally involving dysregulation of multiple signal transduction pathways. In this study, we developed a novel approach to efficiently define dysreguated pathways associated with metastasis by comparing global gene and protein expressions of two distinct metastasis-suppressed models. Consequently, we identified common features shared by the two models which are potentially associated with metastasis. The efficiency of metastasis from the highly aggressive polyoma middle T-induced mouse mammary tumors was suppressed by either prolonged caffeine exposure or by breeding the animal to a low metastatic mouse strain. Molecular profiles of the primary tumors from both metastasis-suppressed classes were then derived to identify molecules and pathways that might underlie a common mechanism of metastasis. A number of differentially regulated genes and proteins were identified, including genes encoding basement membrane components, which were inversely related to metastatic efficiency. In addition, the analysis revealed that the Stat signal transduction pathways were potentially associated with metastasis inhibition, as demonstrated by enhanced Stat1 activation, and decreased Stat5 phosphorylation in both genetic and pharmacological modification models. Tumor cells of low-metastatic genotypes also demonstrated anti-apoptotic properties. The common changes of these pathways in all of the metastasis-suppressed systems suggest that they may be critical components in the metastatic cascade, at least in this model system. Our data demonstrate that analysis of common changes in genes and proteins in a metastatic-related context greatly decrease the complexity of data analysis, and may serve as a screening tool to identify biological important factors from large scale data.     

Genomic Analysis of Stress Response against Arsenic in Caenorhabditis elegans

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.     

Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

Background

The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals.

Results

A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance.

Conclusion

The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity.     

FTSH4 and OMA1 mitochondrial proteases reduce moderate heat stress-induced protein aggregation

The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.

Mitochondrial proteases prevent accumulation of insoluble protein aggregates and protect Arabidopsis plants against long-term moderate heat stress.     

Exploring biomolecular machines: energy landscape control of biological reactions

For almost 15 years, our Pathway model has been the most powerful model in terms of predicting the tunnelling mechanism for electron transfer (ET) in biological systems, particularly proteins. Going beyond the conventional Pathway models, we have generalized our method to understand how protein dynamics modulate not only the Franck-Condon factor, but also the tunnelling matrix element. We have demonstrated that when interference among pathways modulates the electron tunnelling interactions in proteins (particularly destructive interference), dynamical effects are of critical importance. Tunnelling can be controlled by protein conformations that lie far from equilibrium-those that minimize the effect of destructive interference during tunnelling, for example. In the opposite regime, electron tunnelling is mediated by one (or a few) constructively interfering pathway tubes and dynamical effects are modest. This new mechanism for dynamical modulation of the ET rate has been able to explain and/or predict several rates that were later confirmed by experiment. However, thermal fluctuations can also affect these molecular machines in many other ways. For example, we show how global transformations, which control protein functions such as allostery, may involve large-scale motion and possibly partial unfolding during the reaction event.     

Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling

Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (T(b)), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted ((15)N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype.     

Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.     

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress.     

Quantitative proteomics using SILAC-MS identifies N-acetylcysteine-solution-triggered reversal response of renal cell carcinoma cell lines

N-acetylcysteine (NAC), a precursor for glutathione (GSH), causes permeable antioxidation protecting normal cells and disrupting cancer cells. In the present study, we found that a NAC-based medium can trigger a reversal response of human clear cell renal cell carcinoma (ccRCC). To further investigate the action of a NAC-based solution in ccRCC cell lines, 786-O and SN12C were incubated in a serum-free acid medium (low pH) in the presence of 2 mM NAC for 24 hours or in a serum-free medium (normal pH) as the control, and then a phenotypic and proteomic analyses were performed. To determine the reversal occurrence, we tested the phenotypic features associated with cancer cells. Under this premise, a systematic and in-depth analysis of NAC-solution-triggered protein alterations was carried out by quantitative proteomics in both cell lines. Among the paramount protein signature, we identified a large number of proteins associated with cancer features were downregulated, but other proteins in the KEGG pathways associated with recovery of the missing tumorigenicity, such as the p53 pathway and repair pathway, were significantly upregulated. Quantification of notable proteins was validated by messenger RNA (mRNA) and protein levels in the ccRCC cell line. Collectively, our data indicate that the NAC-based solution inhibits human ccRCC cell growth by decreasing cell proliferation and inducing apoptosis, limiting their migration by limiting cell motility and completely changing their metabolic mode. Thus, NAC-based solutions could be used for the prevention or treatment of ccRCC.