Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that ...perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.
► We evaluate the mitochondrial subproteome in genomically unstable cells. ► Significant changes in mitochondrial protein levels are observed in unstable cells. ► Mitochondrial and redox gene expression profiles are altered in unstable cells. ► The protein and gene expression changes may be mediated by epigenetic mechanisms. ► Mitochondrial dysfunction contributes to radiation-induced genomic instability.
Posttranslational modifications such as phosphorylation and ubiquitination serve, independently or together, as gatekeepers of protein transport and turnover in normal and disease physiologies. ...Aberrant protein phosphorylation is one of the defining pathological hallmarks of more than 20 different neurodegenerative disorders, including Alzheimer's disease (AD). The disruption of the phosphorylation of neurotransmitter receptors has been implicated as one of the causal factors of impaired memory function in AD (1-3). Another feature of AD is the aberrant accumulation of proteins that are normally degraded by the ubiquitin proteasome system upon being conjugated to ubiquitin. Thus, elucidating the protein targets of phosphorylation and ubiquitination that can serve as AD biomarkers will aid in the development of effective therapeutic approaches to the treatment of AD. This chapter provides details pertaining to the qualitative and quantitative liquid chromatography tandem mass spectrometry-based analysis of an affinity purified, phosphorylated, and ubiquitinated protein, paired-helical filament tau.
The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from ...nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography–tandem mass spectrometry (geLC–MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the “bait” protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.
Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid β (Aβ) itself generates ROS that can directly damage ...proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Aβ‐mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two‐dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Aβ‐induced oxidative stress in cultured primary cortical mouse neurons. The Golgi‐resident enzyme glucuronyltransferase (GlcAT‐P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK‐1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Aβ, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK‐1 expression. Moreover, in vivo, HNK‐1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Aβ‐mediated oxidation of GlcAT‐P is impairment of its enzymatic function, thereby disrupting HNK‐1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK‐1 modulates synaptic plasticity and is critically involved in memory consolidation.
Healthful physiology can be distinguished from unhealthful physiology by focusing upon how a given signal transduction pathway is shifted as a function of disease. In order to distinguish between ...pathways that contribute to normal versus disease biology, it is necessary to identify components that comprise a protein module. The development of methods that target such differences is essential for the identification, development and validation of biomarkers that can improve the quality of diagnoses and treatment of disease. This review discusses the use of proteomic methods that integrate cell biology, mass spectrometry and bioinformatics, in relation to the analyses of protein signaling modules that are subject to differential phosphorylation. We examine how these methods can be used to distinguish abnormal from normal physiology.
The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed ...receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid β-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid β-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction.
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