The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Citrullination, a novel post-translational modification of elastin, is involved in COPD Pathogenesis.

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. The current study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Label-free liquid chromatography-mass spectrometry comparison of the breast muscle proteome profiles in two fast-growing broilers.

Poultry meat-production is increasing worldwide; leading to the selection of chickens for meat-production that show a fast growth. A label-free quantitative proteomic-approach and Western-blot were applied to investigate the dynamics of muscle protein under rapid growth conditions in two common fast-growing broiler genetic-lines (Ross 508 and AZ Extra Heavy Red-chicken). Muscle exudate from chicken Pectoralis major was used as substrate to unveil the proteome of these genetic-lines. Six-hundred forty-five proteins were identified in total from all samples, and after statistical-analysis 172 proteins were found to be differentially-expressed, clearly distinguishing the two chicken genetic-lines. Several of these differentially-expressed proteins were involved with the proteasome and glycolysis/gluconeogenesis-pathways. Changes in meat-quality traits were also observed, which were reflected in the proteomic-profile. Proteins involved in the ubiquitin-proteasome system were associated with the bigger muscle mass of Ross 508, while phosphoglucomutase 1 was associated with a possible higher capability of AZ Extra Heavy Red-chickens to cope with stressors. This pilot proteomic-approach applied on muscle exudate samples provided key evidence about the pathways and processes underlying these two chicken genetic-lines and their meat-quality parameters. We also identified potential biomarkers that could determine the peculiar production potentials (e.g. breast-growth) of these broilers-lines, which arise from differences in their genetic-backgrounds.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin-containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis.

Label-Free Quantitative Analysis of Pig Liver Proteome after Hepatitis E Virus Infection.

Hepatitis E represents an emerging zoonotic disease caused by the Hepatitis E virus (HEV), for which the main route of transmission is foodborne. In particular, infection in humans has been associated with the consumption of contaminated undercooked meat of pig origin. The aim of this study was to apply comparative proteomics to determine if porcine liver protein profiles could be used to distinguish between pigs seropositive and seronegative for HEV. Preliminarily, an ELISA was used to evaluate the presence of anti-HEV antibodies in the blood serum of 136 animals sent to slaughter. Among the analyzed samples, a seroprevalence of 72.8% was estimated, and it was also possible to identify 10 animals, 5 positive and 5 negative, coming from the same farm. This condition created the basis for the quantitative proteomics comparison between homogeneous animals, in which only the contact with HEV should represent the discriminating factor. The analysis of the proteome in all samples of liver exudate led to the identification of 554 proteins differentially expressed between the two experimental groups, with 293 proteins having greater abundance in positive samples and 261 more represented in negative exudates. The pathway enrichment analysis allowed us to highlight the effect of the interaction between HEV and the host biological system in inducing the potential enrichment of 69 pathways. Among these, carbon metabolism stands out with the involvement of 41 proteins, which were subjected to interactomic analysis. This approach allowed us to focus our attention on three enzymes involved in glycolysis: glucose-6-phosphate isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and fructose-bisphosphate aldolase A (ALDOA). It therefore appears that infection with HEV induced a strengthening of the process, which involves the breakdown of glucose to obtain energy and carbon residues useful for the virus's survival. In conclusion, the label-free LC-MS/MS approach showed effectiveness in highlighting the main differences induced on the porcine liver proteome by the interaction with HEV, providing crucial information in identifying a viral signature on the host metabolism.

Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.