Characterization of LdLIP3 Enzyme Activity from Leishmania donovani

ABSTRACT Lipases catalyze the hydrolysis of fats to form glycerol and fatty acids. Secreted lipases have been implicated as virulence factors in some pathogens. Previously a gene encoding a secretory lipase from the human protozoan pathogen Leishmania donovani was identified and characterized however, experimental evidence to support the biological role of secreted lipases in Leishmania sp. remains to be elucidated. The goal of this project was to express the secretory lipase gene, LdLIP3, from L. donovani and characterize its lipolytic activity. Culture supernatants of L. donovani over-expressing the secretory LdLIP3 enzyme were used to characterize lipolytic activity at several different pHs and temperatures that are biologically relevant in the life cycle of the parasite. At 26°C the optimal lipolytic activity was determined to be pH5.0 with palmitate as the substrate. Similarly, at 37°C the optimal activity was at pH 6.5 with palmitate, and at 42°C, pH 6.0 using stearate as substrate. These results indicate that LdLIP3 has lypolytic activity that could be significant throughout the parasite life cycle. Characterization of the optimal lipolytic activity for the LdLIP3 enzyme should allow further analysis of the physical and biochemical properties of the secreted lipase and to define its possible role in the life cycle of Leishmania sp. INTRODUCTION There are several species of Leishmania which are known to infect humans Leishmania donovani has two distinct life cycle stages, one in the human and the other in the Phlebotomus sp. sandfly vector Lipases are ubiquitous enzymes, either secreted or membrane-bound, that cleave the ester bonds of fats to release free fatty acids and glycerol Leishmania are typically facultative lipid scavengers. As obligate parasites they must salvage these macromolecules from their hosts. For example, during their life cycle these organisms undergo changes in physiology and architectural membrane remodeling METHODS Lipase and Control Samples: The leishmanial expression plasmid, pKSNEO A 20mM 4-MU stock solution was made in dimethylformamide (DMF) and stored at -20°C. This stock solution was diluted to make twelve "known" concentrations of 4- 11 MU standards which ranged from 0µM -250µM. Standards were run on every 96-well plate experiment to generate independent 4-MU standard curves. Stock solutions of the substrates 4-MU palmitate (16C) and 4-MU stearate (18C) were made using DMF as a solvent at a concentration of 6mM and stored at -20°C. At the end of the enzyme incubation period, stop buffers were added to the assays to bring the pH up to 10.5, the optimal fluorescent condition for 4-MU, before being read by the fluorometer Lipase Assay Conditions: McIlvaine's buffer stock solutions of 0.1M citric acid and 0.2M Na2HPO4 were used to produce nine buffers ranging from pH 4.0-8.0 (Shakarian et al 2010). All buffers were filter sterilized and stored at room temperature. Lipase activity assays were carried out in 96-well plates. Each assay plate tested one pH at one temperature with one substrate, and was set up according to Data Analysis: The 4-MU concentration generated in the assay samples were converted from µM 4-MU released to pmol/min/mL of activity using the standard curve. To normalize the samples, the LdLIP3 negative was subtracted from the LdLIP3 sample. Similarly, the pKSNEO negative was subtracted from the pKSNEO sample. The net activity was then calculated by subtracting the net activity of pKSNEO from the net activity of LdLIP3 RESULTS Lipases are a type of esterase which prefers long chain fatty acids as substrates. While lipases and especially secretory lipases are known virulence factors in other organisms In lipase assays carried out at 26°C A net increase in lipolitic activity at 42°

An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non–cell autonomous neurodegeneration

Perturbations of astrocytes trigger neurodegeneration in several diseases, but the glial cell–intrinsic mechanisms that induce neurodegeneration remain poorly understood. We found that a protein complex of α2-Na/K ATPase and α-adducin was enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS). Knockdown of α2-Na/K ATPase or α-adducin in mutant SOD1 astrocytes protected motor neurons from degeneration, including in mutant SOD1 mice in vivo. Heterozygous disruption of the α2-Na/K ATPase gene suppressed degeneration in vivo and increased the lifespan of mutant SOD1 mice. The pharmacological agent digoxin, which inhibits Na/K ATPase activity, protected motor neurons from mutant SOD1 astrocyte–induced degeneration. Notably, α2-Na/K ATPase and α-adducin were upregulated in spinal cord of sporadic and familial ALS patients. Collectively, our findings define chronic activation of the α2-Na/K ATPase/α-adducin complex as a critical glial cell–intrinsic mechanism of non–cell autonomous neurodegeneration, with implications for potential therapies for neurodegenerative diseases