Characterization of cellular stress systems using biological mass spectrometry

In recent years mass spectrometry has become an invaluable tool to address an array of biological questions. The versatility of this experimental approach does not only allow assignment of protein identity and identification of sequence specific modifications, but with the help of particular derivatization techniques facilitates the determination of peptide quantity. Each of these approaches were applied to the following biological projects: The 21 kDa heat stable protein purified from the encysted embryo of Artemia franciscana was characterized by time-of-flight mass spectrometry. De novo sequencing of peptides identified this protein as a group 1 Late embryogenesis abundant (LEA) protein. The amino acid sequence assignment to these peptides allowed amplification of the entire gene sequence from an embryonic cDNA library. This was deposited into the NCBI database (EF656614). The expression of group 1 LEA protein is consistent with and supports a role in desiccation tolerance. In addition, this is a first report describing identification of a group 1 LEA protein in an animal species. A MS-based quantitative analysis was performed in order to analyze relative changes in the dynamic thiol and disulfide states of the redox sensitive protein disulfide isomerase, PDI. PDI cysteine residues were derivatized with an isotope-coded affinity tag (ICAT), thus allowing a direct comparison between the reduced and auto-oxidized forms. Quantitation was based on relative ratios between light and heavy isotopically labeled cysteine containing peptides. The application of the ICAT-labeling approach to PDI related studies, allowed direct assignment of individual cysteine residues and their oxidation status, compared to the previously employed techniques, that only provided information regarding the average number of modified cysteine residues within PDI, not their exact identity. A combination of a phosphopeptide enrichment step and a MS-based approach was utilized to identify three phosphorylation sites on hYVH1, an atypical dual specificity phosphatase that functions as a cell survival factor. With the help of phosphomimetic and non-phosphorylable mutants, we were able to decipher their effect on localization and progression through the cell cycle. Collectively, these studies manifest the power of MS-generated data to influence and guide many different fields ranging from molecular embryology to biochemistry

Efficacy of Chondroprotective Food Supplements Based on Collagen Hydrolysate and Compounds Isolated from Marine Organisms †

Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions