Current Proteomics - Volume 13, Issue 3, 2016

Background: Proteomics in companion animal medicine has been used chiefly, in order to identify proteins, which may be used as biomarkers for early diagnosis of a variety of pathological conditions, as well as to elucidate pathogenesis of various diseases by describing, at molecular level, signal transductions in diseased organs. Objective: The review of some of currently available knowledge in proteomics, as can be applied in companion animal medicine and can be of use to veterinarians active in the field, in order to solve scientific or clinical questions regarding signal transduction. Method: Specific clinical applications of the methodologies are reviewed. Results: Proteomics may be employed in supporting early and accurate diagnosis of leishmaniosis, as well as for the identification of proteins of the causative protozoon, and can also be helpful in cases of neoplastic diseases; proteins identified in blood serum or tears of dogs or cats may be used to support diagnosis of various neoplastic conditions and to monitor treatment and predict future metastases. Proteomics have helped to identify proteins with diagnostic significance in the blood serum of Cavalier King Charles Spaniels with myxomatous mitral valve degeneration and in the bronchoalveolar lavage fluid of West Highland White Terriers with idiopathic pulmonary fibrosis and have also been used for studying the musculoskeletal system of horses, specifically in laminitis, osteoarthritis and osteochondrosis and exertional rhabdomyolysis. The technology has also been applied in various other diseases of dogs and cats (e.g., Leptospira infections, Babesia infections, Golden Retriever muscular dystrophy, degenerative myelopathy, degenerative changes in the brain, azotaemia, periodontitis), as well as in uveitis and endometritis in horses and in staphylococcal infections and tularaemia in rabbits. Conclusion: In the long term, proteomics will contribute to improvements in all facets of companion animal medicine. Mining deeper into the various proteomes and application of new methodological strategies in clinical studies will provide information about disease processes, which will be of benefit to practising veterinarians. Improvement of diagnostic techniques, establishment of prognostic tools and development of vaccines against diseases are key-areas for targeted research.

Amyloid β-peptide (Aβ1-42) is known to be the primary component of the amyloid plaques found in the brain of patients suffering from Alzheimer’s disease (AD). Extensive research has been done in the past with respect to Aβ1-42 peptide and its involvement in AD. However, the initial seed structure responsible for the Aβ1-42 peptide aggregation and the mechanism remains unclear. This may be chiefly attributed to the poor understanding of the processes involved in the Aβ1-42 peptide mis-folding and aggregation. Further, the flexibility and the aggregation propensity involved in the Aβ1-42 monomer, challenges the experimental and theoretical techniques to characterize the seed structure that drives the mis-folding and the subsequent aberrant aggregation. In this study, we employed all atoms fully unrestrained folding molecular dynamics simulation to identify the probable seed structure of Aβ1-42 peptide responsible for the aggregation. The initial linear structure of the Aβ1-42 peptide for the simulation was built from its amino acid sequence using leap module of AMBER. From our molecular dynamics study, we followed the secondary structure development in Aβ1-42 peptide starting from its initial linear structure to its folded 3D structure. We observed the Aβ1-42 peptide to sample diverse conformations which are rich in β-sheets and are stabilized by hydrogen bonding and other non-bonding interactions. The findings of this study shall be helpful in understanding the initial stage of Aβ1-42 peptide aggregation process and can be applicable for the development of therapeutics to cure AD at an early stage.

Background: The fungal genus Aspergillus contains a number of species that are capable of causing a spectrum of diseases in immunocompromised humans. A. fumigatus accounts for approximately 90% of life-threatening infections. Poly –acid ligand i.e. EDTA having potent antifungal activity against A. fumigatus was investigated for its effect on fungal proteins. Objective: The foremost objective of this research to reveal A.fumigatus proteins inhibited in response to polyacid ligand i.e. EDTA. Methods: The proteins profile has been analysed by silver stained gel electrophoresis. The inhibited proteins then subjected to mass spectrophotometer. Further to quantify the results, morphological effect of EDTA (MIC50-19.0 μg/ml) on fungus is studied by Scanning Electron Microscopy (SEM). The toxicity of EDTA was determined by MTT assay. Results: The research identified expression of two protein(s) i.e. 74/86kDa is inhibited. The peptide mass fingerprinting identified these protein(s) bands as Phenyl alanyl t-RNA synhetase alpha subunit PodG and di-peptidyl peptidase. The Scanning electron microscopy showed EDTA inhibited the hyphal growth and also induced striking changes in hyphal morphology (like wrinkled, flattened hyphae with disrupted tips). These cytological effects on hyphal growth and two targeted proteins of A. fumigatus might be responsible for anti- Aspergillus activity of EDTA. Moreover EDTA was found to be non toxic up to 250 μg/ml by MTT assay. Conclusion: Phenyl alanyl t-RNA synhetase alpha subunit PodG and di-peptidyl peptidase might be further studied to use them as target molecules in future or in translational medicine. It can be proceeded for establishing an animal model to test its efficacy (in progress).

Background: Schizophrenia, a multifactorial disorder exhibits diverse neuropathological aberrations with altered protein expression and post translational modifications as surfacing evidence that may also contribute to its pathophysiology. Objective: To investigate the integrated picture of molecular changes and potential alterations in expression of specific glycosylated proteins in different brain regions compared to physiologically normal brains. We aimed to provide more holistic view of three brain regions; and their dynamic cross-talk providing insights into the underlying molecular mechanisms associated with schizophrenia. Methods: Present study entails, differential proteomic analysis of autopsied brain regions of schizophrenic; substantia nigra, cortex, and hippocampus (n=7 each), by using sodium dodecyl sulphate polyacrylamide gel electrophoresis coupled with immunoblot and DIG (digoxigenin) labelling followed by ESI-QTOF MS analysis for validation. Results: We have detected fourteen glycosylated protein components with altered expression among the three studied brain regions, with two contra and a coregulated protein. The 50KDa (Glial fibrillary acidic protein, GFAP) and 84KDa (mitochondrial inner membrane protein) are contra-regulated between substantia nigra and cortex, while T-complex protein 1 subunit zeta of 58KDa is coregulated between substantia nigra and hippocampus. Besides co and contra regulated proteins, remaining proteins with significantly altered glycosylation intensity in the specific brain regions give evidence of their explicit regional function. Additionally, nine proteins commonly appeared nonglycosylated in the three studied brain regions. Conclusion: The characterization of expressed glycoproteins globally and their co and contra regulation could be due to strong regional association of disease associated pathways probably as a result of their modulatory roles following post-translational modification.

Background: Various taxonomic category of the genus Plasmodium are responsible for malaria, the disease induced by P. falciparum is life-threatening if left untreated. The degradation of hemoglobin is a major metabolic process required for the endurance of Plasmodium in its host. Plasmepsin-I is the most promising drug target for malaria. Plasmepsins are the other name for the aspartic proteases of Plasmodium spp., which are necessitated in the degradation of hemoglobin within the food vacuole throughout the erythrocytic stage of the parasite’s life cycle. Objective: Identification of novel inhibitors for plasmepsins through virtual screening, docking and simulation. Methodology: We generated diverse combinatorial library-based ligands to achieve this aim. Structures for 11826 lead and drug like molecules were downloaded from the zinc database in mol2 format for known inhibitors of plasmepsin protein, using code 3QRV, and were used for virtual screening with Openeye Scientific software. Various analysis were carried out to study the consequences of this virtual screening and docking approach, including residue interactions with key residues of the protein, Chemgauss4 scores, shape, hydrogen bonding, and binding energy. Results/Conclusion: Our work demonstrates not just the relevancy of computer aided drug discovery, merely as well places a range of small molecules that hold the potential to serve as candidates for inhibition of P. falciparum plasmepsins. Utilizing the approach of virtual screening, Molecular Docking, Molecular dynamics simulation and ADME/T versus the malaria-causing parasite P. falciparum, we demonstrate that a computer-aided drug design infrastructure provides new models to explore battle against disease of the inadequate. The results describe a group of top-ranking compounds for further consideration.

Background: Amniotic fluid is a complex mixture and reflects the physiological status of the developing fetus. Many proteins presented in amniotic fluid are of exceptional interest because their expression reflects physiological and pathological conditions of the fetus and/or pregnancy. Objective: In the present study, we performed proteomics-based identification of biomarkers of amniotic fluid from normal and pathological pregnancy. Methods: Proteins isolated from amniotic fluid of three different status of pregnancy - normal, preeclampsia and polyhydramnios, were fractionated by 2DE, visualized by Coomassie blue staining and all visualized proteins subjected for identification with mass spectrometry. Around 40 proteins were identified by mass spectrometry analysis. Changes in identified protein levels defined in three different pregnancy status were evaluated by using computational methods. Results: In our study we characterized proteins isolated from amniotic fluid obtained from normal pregnancy, developed preeclampsia and polyhydramnios. Some proteins’ are variable in their expression level in comparison to pathological cases with normal pregnancy. These proteins are involved in various cellular processes and are responsible for cell signaling and regulation (apolipoprotein, peroxiredoxin, protein AMBP, putative elongation factor, clusterin, etc.), metabolic processes (lumican, vitamin D binding protein, etc.), macromolecular transport (ceruloplasmin, corticosteroidbinding protein, etc.), proteins specific for pregnancy and embryo development (angiotensinogen, fibrinogen beta chain, etc.) and others. Conclusion: In the study new proteins typical for amniotic fluid of preeclampsia and polyhydramnios pregnancies were identified. These proteins are associated with development, signal transduction, and metabolic processes. Results could be valuable for developing biomarkers for fetal development and pregnancy-related disorders.

Drought is a major environmental factor that limits grain yield in rice. In order to understand the proteomic changes in response to drought at reproductive stage, proteins expressed in the young panicles were compared between two varieties of rice with contrasting tolernace to drought. The identified 36 differentially expressed proteins are involved in energy production, metabolism, stress defense, protein destination and cell structure. A coordinative up-regulation of triosephosphate isomerase, glycin decarboxylase complex H-protein, glyoxylase I and cytosolic ascorbate peroxidase was obseved in the drought tolerant variety Zhonghan 3, which may contribute to higher tolerance by increasing the energy production and maintaining redox homeostasis. Two-dimensional Western blotting experiments identified three APX1 and three APX2 isoforms up-regulated during drought stress in drought-tolerant varieties (Zhonghan 3, IRAT109 and PRATAO). However, in droughtsuceptible varieties (IR29, Zhengshan 97B and CATUNA), expression of the six isoforms was unchanged or even down-regulated. Under normal growth conditions, expression level of the six APX isoforms showed negative correlation with drought tolerance of rice. Our data suggested that APX isoforms may be considered as indicators of the redox status that related to drought tolerance in rice.

Type 2 diabetes mellitus is characterized by hyperglycemia and insulin resistance. Liver is a key organ for the maintenance of systemic glucose homeostasis in mammals, including humans and mice. Under many nutritional conditions the liver maintains nearly constant blood glucose. We analyzed the protein profile from the liver from ten-week old diabetic obese db/db mice through twodimensional electrophoresis (2DE) and the identity of differentially expressed proteins was determined by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Some of these proteins were pyruvate carboxylase, carbamoyl-phosphate synthase, succinate dehydrogenase, ornithine aminotransferase, selenium-binding protein, proteasome subunit alpha type-2, regucalcin, creatine kinase M-type, and others, which are involved in metabolic process of carbohydrates, lipids and proteins. Results suggest that alteration in the expression of these proteins plays a role in the pathogenesis of obesity and diabetes. In addition, we identified some proteins that have not been previously associated with type 2 diabetes mellitus.

SILAC (Stable Isotope Labeling with Amino acids in Cell culture) is one of the most popular methods in quantitative proteomics field. The internal standards of SILAC can mark a variety of clinical samples or tissues instead of labeling the whole organism and play an important role in proteomics. Preparing the SILAC internal standards has been difficult and time-consuming for culturing cell lines, therefore a simple and convenient Re-SILAC method was introduced. Cell lines labeled with SILAC were thawed in SILAC medium to be remarked again. Comparing the morphology, size, growth curve and proteome of MHCC97-L between Re-SILAC and SILAC cell lines, it was found that SILAC-labeled cells that were frozen with liquid nitrogen had no influence on Re- SILAC-labeled. In conclusion, Re-SILAC method is a useful method for preparing SILAC internal standards.