Identification of Hypoxia-Induced Alternative Splicing Using Proteomics Approach
Abstract
Alternative splicing is a regulatory process for gene expression, which results in various protein products from one single gene. It greatly enriches biodiversity, especially when cells are under environmental stress like hypoxia. Moreover, some abnormal splicing variations are also implicated in diseases. Genome-wide analysis of alternative splicing is typically pursued on the genome level using high-throughput approaches such as DNA microarray or deep sequencing. However, these methods examine the alternatively spliced transcripts instead of the protein products. In the current study, a novel proteomics approach was developed to confirm the existence of alternative spliced proteins by mass spectrometry. Since the outgrown cancer cells are under hypoxia stress, alternative splicing is implicated under such harsh condition. Therefore, the resultant cancer-specific splicing variants may potentially serve as diagnostic, prognostic, or predictive biomarkers. We have identified a few novel alternative spliced variants using this new approach and the consequent biological evaluation is undergoing. We are convinced that this method will provide a new approach for the study of alternative splicing in the future.
Phosphoproteomic profiling of ERK MAP kinase signaling reveals a role of phosphorylation in the interaction of nucleoporins with transport factors
Abstract
Many ERK MAP kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. We have developed a phosphoproteomic approach combining use of the steroid receptor fusion system, IMAC, two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), and phosphomotif-specific antibodies, and applied this system for the global identification of ERK substrates. Quantitative analysis detected 37 reproducibly changed spots between ERK-activated and -inhibited samples, and 24 identified proteins were considered novel candidates for ERK targets, which suggests as yet undefined roles for this signaling pathway in various physiological processes. We confirmed the phosphorylation of these candidate substrates in ERK-activated cells by 2-D western blotting, and observed that ERK directly phosphorylated at least 13 proteins in vitro.
Of these, the nucleoporin Nup50 was verified as a bona fide ERK substrate. Interestingly, ERK phosphorylation of the FG repeat region of Nup50 was found to reduce its affinity for importin-β family proteins, importin-β and transportin. Other FG nucleoporins such as Nup153 and Nup214 also showed a similar functional change after ERK-mediated phosphorylation. Furthermore, nuclear migration of importin-β and transportin was impaired in ERK-activated, digitonin-permeabilized cells, and ERK phosphorylation of Nup50 was involved in this impairment. Thus, we propose that ERK may phosphorylate various nucleoporins to regulate nucleocytoplasmic transport.
Our system can be applied to various kinases, and in fact, we have succeeded in identifying new downstream substrates of GRK6 and PKD2/3. These results will also be presented. Our challenges for the next step would be to collectively identify the targets of a given kinase that causes human disease. Global identification of all physiologically relevant phosphorylation sites, and to describe the difference between the phophoproteomes of normal and pathological states, will greatly facilitate the process.
Applications of bacterial proteomics and mass spectrometry to laboratory medicine
Abstract
The majority of clinical decision making is based on labaratoty test results. At present, most routine clinical chemistry tests take advanatage of spectrophotometric or immunoassay-based methods. Progress in mass spectrometry (MS) has significantly contributed to proteome-based biomarker discovery and also to applications of MS to laboratory medicine. MS is a powerful analytical tool used in an increasing number of clinical laboratories around the world. MALDI-TOF MS has proven to be a rapid and reliable tool for identifying microorganisms, including bacteria, yeast, molds, and mycobacteria. In this presentation, first, I am going to talk about the state of arts of proteome-based bacterial identification. Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Until recently, clinical microbiology laboratories relied heavily on conventional methods that often initially involve culturing, followed by morphologic phenotyping and cumbersome biochemical testing. These procedures are time-consuming, laborious, and require well-trained technicians for correct interpretation of the results.Genome-based diagnosis of infectious diseases is promising, but still not simple enough for routine use. By contrast, proteome analysis of bacterial proteins by MALDI-TOF MS is a quick, simple and reliable method for identification of microorganisms. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. A small portion of a cultured bacterial colony is directly subjected to MALDI-TOF MS. The final mass spectral signature is composed of peaks originating from bacterial proteins including mainly ribosomal proteins. The spectral profiles are compared with a library of known spectra and a result is generated within 10 min instead of almost one day when conducted by the traditional methods. Using MALDI Biotyper system (Bruker Daltonics), the rate of successful identification at the species and genus levels in our hands were 91.7% and 97 %, respectively, that is not necessarily satisfactory. One of the reasons for the problematic identifications may be incomplete databases. We have found that refinement of a commercial database can be achieved relatively easily and effectively by incorporating MS spectra of clinical isolates obtained in each clinical laboratory to reduce problematic identifications. Direct analysis of clinical samples without the need for prior culturing might shorten the time required for identifying microorganisms and would therefore further increase the usefulness of the MALDI-TOF MS–based approach. Progress has been made for direct analysis of bacterias in three types of specimens; urine, cerebrospinal fluid (CSF), and blood. In addition to identification of bacteria, other uses of MS in clinical microbiologyu are being actively investigated most importantly for detection of antibiotic resistance. For this purpose, LC-MS/MS could be useful as well. LC-MS/MS has been increasingly used for newborn screening, toxicology, therapeutic drug monitoring, endocrinology, and more recently for measurement of targeted proteins and peptides. Although immunoassay-based methods have dominated steroid hormone measureuents for many years, LC-MS/MS offers significant advantages over immunoassays in terms of analytical sensitivity and specificity. One of the major problems in immunoassays for steroid hormones is cross reactivity. To avoid these problems, LC-MS/MS is increasingly used. As the second topic of this talk, I am going to show some our own data indicating advantages of LC-MS/MS over immunoassays. As a matter of fact, the editorial board of the Journal of Clinical Endocrinology and Metabolism stated in 2015 that “manuscripts reporting sex steroid assays as important endopoint must use MS-based assays”. Although still controversial, it is very likely that this statement will extend to other analytes as well in the near future.
Huazhong University of Science and Technology, China
Title
PTM Bioinformatics: from phosphoproteomics to phosphoproteogenomics
Abstract
Phosphorylation plays essential roles in the regulation of almost all of biological processes and cellular signaling pathways. Recent progresses in phosphoproteomics have identified over 500,000 phosphorylation sites. However, how to efficiently retrieve useful information from flood of data is still a great challenge. To integrate both large-scale and small-scale experimental data, we developed a number of databases, such as dbPPT containing 82,175 phosphorylation sites (p-sites) in 31,012 proteins for 20 plant species (http://dbppt.biocuckoo.org/), dbPSP containing 7,391 p-sites in 3,750 prokaryotic proteins (http://dbpsp.biocuckoo.org/), and dbPAF containing 483,001 p-sites of 54,148 proteins for human, animals and fungi (http://dbpaf.biocuckoo.org/). To predict potentially site-specific kinase-substrate relations (ssKSRs) or kinase-specific p-sites, we developed an algorithm of Group-based Prediction System (GPS), which provides both online service and local packages at http://gps.biocuckoo.org/, and can predict ssKSRs for 408 human protein kinases. In addition, to greatly reduce false positive predictions, we integrated protein-protein interactions between kinases and substrates and further developed the in vivo GPS (iGPS, http://igps.biocuckoo.org/) tool for the prediction of ssKSRs. Using these databases and tools, we first analyzed the phosphorylation regulation in neuronal autophagy, which has become a promising therapeutic target against neurodegenerative diseases. Here, quantitative phosphoproteome profiling together with computational analysis was performed to delineate the distinct phosphorylation signaling networks regulated by two natural neuroprotective autophagy enhancers. We totally quantified >5,000 unique p-sites in over 2,300 phosphoproteins. To identify key regulators, namely protein kinases, we developed a novel network-based algorithm of in silico Kinome Activity Profiling (iKAP) to computationally infer the protein kinase activities from phosphorylation networks. Using this algorithm, we observed that distinct kinases were differentially regulated by the two small molecules. We predicted and validated a well-documented autophagy kinase, p70S6K, to be down-regulated exclusively by one of the two enhancers. We also discovered several new regulators that potentially participate in autophagy, and the inhibition of kinase activities will inhibit autophagy and dramatically diminish the enhancer-mediated clearance of Alzheimer's disease-associated β-amyloid precursor protein and Parkinson's disease-associated α-synuclein. Besides the computational analyses of phosphoproteomics data, we further analyzed genetic variations (GVs) that potentially reconfigure the phosphorylation network, and computationally detected 9606 potential phosSNPs (phosphorylation-related single nucleotide polymorphisms), including 720 known, disease-associated SNPs that dramatically modify the human phosSNP-associated kinase-substrate network. Further analyses demonstrated that the proteins in the network are heavily associated in various signaling and cancer pathways, while cancer genes and drug targets are significantly enriched. We re-constructed four population-specific kinase-substrate networks and found that several inherited disease or cancer genes, such as IRS1, RAF1, and EGFR, were differentially regulated by phosSNPs. Thus, phosSNPs may influence disease susceptibility and be involved in cancer development by reconfiguring phosphorylation networks in different populations. Moreover, by systematically characterizing potential phosphorylation-related cancer mutations (phosCMs) in 12 types of cancers, we observed that both types of GVs preferentially occur in the known cancer genes, while a considerable number of phosphorylated proteins, especially those over-representing cancer genes, contain both phosSNPs and phosCMs. Furthermore, it was observed that phosSNPs were significantly enriched in amplification genes identified from breast cancers and tyrosine kinase circuits of lung cancers. Taken together, our studies not only generated useful resources and tools for the analysis of phosphoproteomic data, but also provided an efficient approach to interpret genomic sequencing data.
Proteomic-scale approaches for identifying reversible and irreversible cysteine redox post-translational modifications in myocardial ischemia/reperfusion
Abstract
Redox post-translational modifications (PTM) are emerging as important regulatory mechanisms in signaling and pathogenesis. Cysteine (Cys) is the most redox active amino acid and is a target for these PTM, some of which are biologically reversible (e.g. disulfides, S-nitrosylation, S-glutathionylation) while others (sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) are considered irreversible. We have developed enrichment methods to examine these PTM on a proteome-wide scale. Rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified by tandem mass spectrometry (MS/MS). We next developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. ‘negative’ selection) followed by ‘positive’ selection using hydrophilic interaction liquid chromatography (HILIC). We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 µM) or to ischemia / reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair. Finally, we have combined these methods to enable multiplexed quantitative analysis of reversible/irreversible Cys redox PTM in response to I/R and in the presence of a thiol-based antioxidant (N-2-mercaptopropionyl glycine, MPG). We quantified >1350 Cys sites that are reversibly oxidized and 215 that are irreversibly oxidized by I/R, including many sites that are protected by MPG. This technique allows for the quantitative profiling of reversible/irreversible Cys PTMs in response to oxidant / antioxidant stimulus, and their delineation within the context of protein abundance, during I/R injury and cardioprotection.
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