Identification of protein carbonylation sites by two-dimensional liquid chromatography in combination with MALDI- and ESI-MS

Oxidative stress is defined as excessive production of reactive oxygen species (ROS) overwhelming the cellular antioxidant defense systems and thereby damaging most constituents of cells including proteins. Reactive carbonyls, i.e. aldehydes, ketones and lactams, are a major class of irreversible oxidative protein modifications that are widely used as biomarkers of oxidative stress, aging and age-related diseases. Whereas carbonylated proteins can be studied by western blotting and ELISA, their site specific mapping still remains a challenging task due to their low abundance and insufficient ionization. Here, we present a new strategy to identify carbonylation sites in a bottom-up approach. Protein digests were derivatized with 2,4-dinitrophenyl hydrazine (DNPH) and separated by hydrophilic interaction chromatography (HILIC). Peptide-containing fractions were then analyzed by laser-desorption/ionization with DNPH as the reactive matrix, which favors DNP-labeled peptides. The mass list generated for each HILIC fraction, representing mostly DNP-modified peptides, was used in the subsequent nano reversed-phase chromatography (RPC) coupled on-line to an electrospray ionization Orbitrap mass spectrometer recording the tandem mass spectra in data dependent acquisition mode. This comprehensive two-dimensional HILIC×RPC-strategy was exemplified for tryptic digests of native bovine serum albumin (BSA) and β-lactoglobulin (β-LG), as well as their in vitro oxidized versions, i.e. oxBSA and oxβ-LG. In total, three carbonylation sites were identified in native β-LG, nine in native BSA, eleven in oxβ-LG and 32 in oxBSA.     

Human and mouse mutations in WDR35 cause short-rib polydactyly syndromes due to abnormal ciliogenesis

Defects in cilia formation and function result in a range of human skeletal and visceral abnormalities. Mutations in several genes have been identified to cause a proportion of these disorders, some of which display genetic (locus) heterogeneity. Mouse models are valuable for dissecting the function of these genes, as well as for more detailed analysis of the underlying developmental defects. The short-rib polydactyly (SRP) group of disorders are among the most severe human phenotypes caused by cilia dysfunction. We mapped the disease locus from two siblings affected by a severe form of SRP to 2p24, where we identified an in-frame homozygous deletion of exon 5 in WDR35. We subsequently found compound heterozygous missense and nonsense mutations in WDR35 in an independent second case with a similar, severe SRP phenotype. In a mouse mutation screen for developmental phenotypes, we identified a mutation in Wdr35 as the cause of midgestation lethality, with abnormalities characteristic of defects in the Hedgehog signaling pathway. We show that endogenous WDR35 localizes to cilia and centrosomes throughout the developing embryo and that human and mouse fibroblasts lacking the protein fail to produce cilia. Through structural modeling, we show that WDR35 has strong homology to the COPI coatamers involved in vesicular trafficking and that human SRP mutations affect key structural elements in WDR35. Our report expands, and sheds new light on, the pathogenesis of the SRP spectrum of ciliopathies.     

From components to regulatory motifs in signalling networks.

The developments in biochemistry and molecular biology over the past 30 years have produced an impressive parts list of cellular components. It has become increasingly clear that we need to understand how components come together to form systems. One area where this approach has been growing is cell signalling research. Here, instead of focusing on individual or small groups of signalling proteins, researchers are now using a more holistic perspective. This approach attempts to view how many components are working together in concert to process information and to orchestrate cellular phenotypic changes. Additionally, the advancements in experimental techniques to measure and visualize many cellular components at once gradually grow in diversity and accuracy. The multivariate data, produced by experiments, introduce new and exciting challenges for computational biologists, who develop models of cellular systems made up of interacting cellular components. The integration of high-throughput experimental results and information from legacy literature is expected to produce computational models that would rapidly enhance our understanding of the detail workings of mammalian cells.     

A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min⁻¹ mg⁻¹) as compared to unmodified pH tuned lipase (128 nmol min⁻¹ mg⁻¹). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min⁻¹ mg⁻¹, respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content.     

Insights into Fanconi Anaemia from the structure of human FANCE

Fanconi Anaemia (FA) is a cancer predisposition disorder characterized by spontaneous chromosome breakage and high cellular sensitivity to genotoxic agents. In response to DNA damage, a multi-subunit assembly of FA proteins, the FA core complex, monoubiquitinates the downstream FANCD2 protein. The FANCE protein plays an essential role in the FA process of DNA repair as the FANCD2-binding component of the FA core complex. Here we report a crystallographic and biological study of human FANCE. The first structure of a FA protein reveals the presence of a repeated helical motif that provides a template for the structural rationalization of other proteins defective in Fanconi Anaemia. The portion of FANCE defined by our crystallographic analysis is sufficient for interaction with FANCD2, yielding structural information into the mode of FANCD2 recruitment to the FA core complex. Disease-associated mutations disrupt the FANCE–FANCD2 interaction, providing structural insight into the molecular mechanisms of FA pathogenesis.     

A Time-Series-Based Feature Extraction Approach for Prediction of Protein Structural Class

This paper presents a novel feature vector based on physicochemical property of amino acids for prediction protein structural classes. The proposed method is divided into three different stages. First, a discrete time series representation to protein sequences using physicochemical scale is provided. Later on, a wavelet-based time-series technique is proposed for extracting features from mapped amino acid sequence and a fixed length feature vector for classification is constructed. The proposed feature space summarizes the variance information of ten different biological properties of amino acids. Finally, an optimized support vector machine model is constructed for prediction of each protein structural class. The proposed approach is evaluated using leave-one-out cross-validation tests on two standard datasets. Comparison of our result with existing approaches shows that overall accuracy achieved by our approach is better than exiting methods.