The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

Cell-penetrating peptides : Uptake mechanism and the role of receptors

Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p

Understanding the dual nature of lysozyme: part villain – part hero : A Drosophila melanogaster model of lysozyme amyloidosis

Amyloid proteins are a distinct class of proteins that can misfold into β-sheet rich structures that later mature to form the characteristic species known as amyloid fibrils, and accumulate in tissues in the human body. The misfolding event is often caused by mutations (or outer factors such as changes in pH) that destabilize the native protein structure. The mature amyloid fibrils were initially believed to be associated with diseases connected to protein misfolding such as Alzheimer’s disease (AD), Parkinson’s disease, transthyretin amyloidosis and lysozyme amyloidosis. However, now it is known that many different factors are involved in these diseases such as failure in protein clearance, lysosomal dysfunction and formation of intermediate misfolded protein species, which possess cytotoxic properties, preceding the formation of mature fibrils. In this thesis the amyloidogenic protein lysozyme has been examined in vivo by using Drosophila melanogaster (fruit fly) as a model organism. The effects of over-expressing human lysozyme and amyloidogenic variants in Drosophila have been investigated both in the absence and presence of the serum amyloid P component (SAP), a protein known to interact with amyloid species. In addition, the role of lysozyme in AD has been investigated by  co-expressing human lysozyme and amyloid β in Drosophila. The lysozyme protein is an enzyme naturally found in bodily fluids such as tears, breast milk and saliva. It is engaged in the body’s defense and acts by hydrolyzing the cell wall of invading bacteria. Certain disease-associated point mutations in the gene encoding lysozyme destabilize the protein and cause it to misfold which results in systemic amyloidosis. To investigate the in vivo misfolding behavior of lysozyme we developed and established a Drosophila model of lysozyme amyloidosis. SAP is commonly found attached to amyloid deposits in the body; however, the role of SAP in amyloid diseases is unknown. To investigate the effect of SAP in lysozyme misfolding, these two proteins were co-expressed in Drosophila. The amyloid β peptide is involved in AD, building up the plaques found in AD patient brains. These plaques trigger neuroinflammation and since lysozyme is upregulated during various inflammation conditions, a possible role of lysozyme in AD was investigated by overexpressing lysozyme in a Drosophila model of AD. Interaction between lysozyme and the amyloid β protein was also studied by biophysical measurements. During my work with this thesis, the dual nature of lysozyme emerged; on the one hand a villain, twisted by mutations, causing the lysozyme amyloidosis disease. On the other hand a hero, delaying the toxicity and maybe the neurological damage caused by the amyloid β peptide

Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.Funding Agencies|Swedish Research Council; Soderberg foundation [M26/11]; Linkoping University Neurobiology Center</p

Comparison of three methods for single-turnover kinetics of two DNAzymes, DzSJ and Dz451.

<p>Kinetic analysis of DzSJ (<b>A</b>, <b>C</b> and <b>E</b>) and Dz451 (<b>B</b>, <b>D</b> and <b>F</b>) by three different methods: conventional gel assay (<b>A</b> and <b>B</b>), EtBr plate assay (<b>C</b> and <b>E</b>) and PicoGreen plate assay (<b>D</b> and <b>F</b>). The fitted k_obs-value for decay curves are shown as min^-1 inserted in each respective graph. Blue dots indicate DNAzyme with substrate, green dots control wells with EDTA and brown dots inactive DNAzyme controls. Orange line represents one phase decay fitting. Data shown as fluorescence mean normalized to initial data-point and control wells without RNA target ± SEM of three independent experiments, each performed in triplicate.</p

Synthetic oligonucleotides.

<p>DNAzymes, RNA substrates and PCR primers for reverse transcription. Underlined letters denote ‘10–23’ catalytic loop of DNAzymes; Bold letters are inactivating G-C mutations in catalytic loop; Italics are ribonucleotides; | represent the cleavage site.</p><p>Synthetic oligonucleotides.</p

The presence of SAP prevents neuronal cell death in F57I-expressing flies.

<p>(A) Apoptotic cells in control, WT and F57I, with and without co-expressing SAP, identified by TUNEL staining at day 0 and day 25. Micrographs were taken at 40x magnification. (B) The number of TUNEL-positive cells in the brains of F57I-expressing flies, collected at day 25, compared with the number of TUNEL-positive cells in age-matched control, WT, control-SAP, WT-SAP and F57I-SAP flies. Bars represent means ± s.e.m, n = 3 brain sections (*p<0.05).</p

Co-expression of SAP does not prevent UPR activation induced by F57I expression in the fly CNS.

<p>(A) Sections (10 μm) of <i>Drosophila</i> brains at day 10 stained with anti-EGFP antibody, then counterstained with DAPI. Red stain shows EGFP-positive cells and blue stain corresponds to the cell nuclei. White arrows indicate EGFP-positive cells. (B) Scatter plot shows number of EGFP-positive cells where the micrographs were examined in a blinded experiment, n = 12, 12, 15, 10, 10 and 12 for Control, WT, F57I, Control-SAP, WT-SAP and F57I-SAP, respectively (*p<0.05, **p<0.01). Scale bars = 50 μm.</p

Comparison of k_obs-values of each DNAzyme with respective target RNA for each kinetic assay.

<p>Data is presented as k_obs ± standard error of the mean. n.d. = not determined.</p><p>Comparison of k_obs-values of each DNAzyme with respective target RNA for each kinetic assay.</p