Evolutionary analysis of enzymes using Chisel

MOTIVATION: Availability of large volumes of genomic and enzymatic data for taxonomically and phenotypically diverse organisms allows for exploration of the adaptive mechanisms that led to diversification of enzymatic functions. We present Chisel, a computational framework and a pipeline for an automated, high-resolution analysis of evolutionary variations of enzymes. Chisel allows automatic as well as interactive identification, and characterization of enzymatic sequences. Such knowledge can be utilized for comparative genomics, microbial diagnostics, metabolic engineering, drug design and analysis of metagenomes. RESULTS: Chisel is a comprehensive resource that contains 8575 clusters and subsequent computational models specific for 939 distinct enzymatic functions and, when data is sufficient, their taxonomic variations. Application of Chisel to identification of enzymatic sequences in newly sequenced genomes, analysis of organism-specific metabolic networks, 'binning' of metagenomes and other biological problems are presented. We also provide a thorough analysis of Chisel performance with other similar resources and manual annotations on Shewanella oneidensis MR1 genome.     

Identification of sialylated glycoproteins from metabolically oligosaccharide engineered pancreatic cells

In this study, we investigated the use of metabolic oligosaccharide engineering and bio-orthogonal ligation reactions combined with lectin microarray and mass spectrometry to analyze sialoglycoproteins in the SW1990 human pancreatic cancer line. Specifically, cells were treated with the azido N-acetylmannosamine analog, 1,3,4-Bu₃ManNAz, to label sialoglycoproteins with azide-modified sialic acids. The metabolically labeled sialoglyproteins were then biotinylated via the Staudinger ligation, and sialoglycopeptides containing azido-sialic acid glycans were immobilized to a solid support. The peptides linked to metabolically labeled sialylated glycans were then released from sialoglycopeptides and analyzed by mass spectrometry; in parallel, the glycans from azido-sialoglycoproteins were characterized by lectin microarrays. This method identified 75 unique N-glycosite-containing peptides from 55 different metabolically labeled sialoglycoproteins of which 42 were previously linked to cancer in the literature. A comparison of two of these glycoproteins, LAMP1 and ORP150, in histological tumor samples showed overexpression of these proteins in the cancerous tissue demonstrating that our approach constitutes a viable strategy to identify and discover sialoglycoproteins associated with cancer, which can serve as biomarkers for cancer diagnosis or targets for therapy.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9083-8) contains supplementary material, which is available to authorized users.     

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.Androgen is important for the development, function, and proliferation of both normal and cancerous prostate cells (1). At the earliest stage of prostate cancer, prostate cancer cells are dependent on the presence of androgen, and androgen-deprivation therapy (ADT)¹ is used to treat prostate cancer (2). However, cells become androgen-independent as a result of androgen deprivation therapy, and they become more aggressive. This results in androgen-independent remission of prostate cancer (3). LNCaP and PC3 cell lines have been widely used as models of prostate cancer. LNCaP is an androgen-dependent cancer cell line, whereas PC3 is an androgen-independent cell line. The LNCaP cell line is less aggressive as compared with PC3 cells that have a high metastatic potential. LNCaP and PC3 cells have been previously studied by genomics and proteomics approaches to understand the mechanism(s) responsible for the aggressive and metastatic nature of prostate cancer (4–8).Post-translational modifications (PTMs) such as phosphorylation are important in the function of the androgen-dependent pathway. Androgen receptors bind to androgen and are then phosphorylated before translocating into the nucleus (3). However, protein PTMs cannot be directly inferred from gene expression. Glycosylation is an abundant PTM and most cell surface or secreted proteins are expected to be glycosylated (9). Glycosylation is one of the more complex PTMs because of the fact that different glycosylation machineries are present in different cells, multiple glycosylation sites exist on many glycoproteins and each glycosylation site can be modified by several different glycans (10, 11). Such microheterogeneity of glycan structures at each glycosylation site with different site occupancy significantly increases the structural diversity of each glycoprotein that is specific to the microenvironment of the cells where each glycoprotein is produced. Although these characteristics of protein glycosylation pose considerable challenges to the structural and functional analyses of glycoproteins, we expect that cell and cell microenvironment-specific glycoproteins differ according to the physiological and pathological states of the cells. Aberrant glycosylation is the result of alterations in glycosylation genes that may lead to the development of cancer. A systematic approach to analyze proteins, glycoproteins, and glycosylation is expected to permit the identification of the glycoprotein alterations that are specific to each cell state and aid the understanding of the functions of glycosylation because alterations in glycosylation can affect glycoprotein abundance or function (12, 13). A detailed analysis of glycoproteins in cancer cells with different functions is needed to understand tumor biology and how glycoproteins can function as therapeutic targets or diagnostic biomarkers (14, 15).In this study, a comprehensive proteomic and glycoproteomic platform was designed to investigate the differences in proteins, glycoproteins, and site-specific glycosylation forms of glycoproteins between LNCaP and PC3 cells (Fig. 1). To our knowledge, this is the first report to characterize glycoproteins with respect to protein abundance, glycosylation occupancy, and glycosylation heterogeneity at specific glycosites. These altered glycosylation patterns among proteins between LNCaP and PC3 cell lines have a significant potential to aid our understanding of the altered glycoprotein expression in prostate cancer cells, thus leading to novel specific methods to detect aggressive prostate cancer.Open in a separate windowFig. 1.Schematic representation of the workflow for the integrated analysis of glycosite-containing peptides, global protein expression, and intact glycopeptides. Proteins were obtained from LNCaP and PC3 cell lines followed by tryptic digestion and iTRAQ labeling. Labeled peptide samples were then combined and separated into two aliquots. One aliquot was enriched for glycosite-containing peptides using Solid Phase Extraction of Glycopeptides (SPEG) and the other aliquot was used for bRPLC separation followed by the analysis of global proteins and intact glycopeptides. Finally, peptides were analyzed using LC-MS/MS.     

Strategies of Bringing Drug Product Marketing Applications to Meet Current Regulatory Standards

In the past decade, many guidance documents have been issued through collaboration of global organizations and regulatory authorities. Most of these are applicable to new products, but there is a risk that currently marketed products will not meet the new compliance standards during audits and inspections while companies continue to make changes through the product life cycle for continuous improvement or market demands. This discussion presents different strategies to bringing drug product marketing applications to meet current and emerging standards. It also discusses stability and method designs to meet process validation and global development efforts.At the 2014 American Association of Pharmaceutical Scientists (AAPS) annual meeting in San Diego, CA, Yan Wu (Merck) and Anita Freed (Pfizer) led a symposium entitled “Bringing Drug Product Marketing Applications to Current Regulatory Standards: Trials and Tribulations.” This symposium was very timely as this topic is a growing industry concern, evidenced by over 300 attendees, and considering the new guidances (1–8) that have been established over the past decade. While most of these quality standards are applicable to new drug products, there is a risk that currently marketed products, known as legacy products, will not meet the new compliance standards during audits and inspections. Companies also need to continuously make process or method changes for in-line products as part of product life cycle management efforts or to meet different market needs. If legacy (or in-line) products undergo a change, the question is how much extra effort is needed to have these products meet current standards to support the associated submission. This symposium addressed these issues and offered modeling tools using existing data or other approaches and case studies to effectively manage post-approval changes. Presentations included the following:

• Modeling historical data to support process and method stability changes
• Food and Drug Administration (FDA) perspectives on application of International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q8 to legacy products
• Assessment of impact on stability with manufacturing, packaging, and/or method changes
• Applying Association of Southeast Asian Nations (ASEAN) stability requirements to legacy products and managing specifications across climatic zones

This paper provides an overview of the presentations and highlights strategies and points of consideration when bringing marketing applications of the legacy drug products to current and emerging standards.     

Modifications of membrane lipids in response to wounding of Arabidopsis thaliana leaves

Mechanical wounding of Arabidopsis thaliana leaves results in modifications of most membrane lipids within 6 hours. Here, we discuss the lipid changes, their underlying biochemistry, and possible relationships among activated pathways. New evidence is presented supporting the role of the processive galactosylating enzyme SENSITIVE TO FREEZING2 in the wounding response.     

Arsenic and Heavy Metal Concentrations in Drinking Water in Pakistan and Risk Assessment: A Case Study

The present study was performed to assess drinking water quality and potential health risk in the Nowshera District, Khyber Pakhtunkhwa, Pakistan. For this purpose drinking water samples were collected from local available sources and analyzed for physico-chemical characteristics, arsenic (As) and heavy metals. Results revealed high levels of toxic heavy metals such as chromium (Cr), nickel (Ni), lead (Pb), cadmium (Cd), and As contaminations in the drinking water. Results were evaluated for chronic risk including average daily intake (ADI) and hazard quotient (HQ). Among heavy metals the HQ values were highest for Cd (5.80) and As (2.00). Therefore, populations in the study area may be at a low level of chronic toxicity and carcinogenic risk. Statistical analyses showed that contribution of different drinking water sources to the mean contaminant levels in the study area was insignificant (p =.53). Correlation analysis further revealed that anthropogenic activities were the main sources of contamination, rather than geogenic. This study strongly recommends the treatment of urban and industrial wastewater in the vicinity of the study area and provision of safe drinking water.     

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ～20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.     

Proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus RNA replication: an inhibitory role of protein kinase C

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.