?2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ?2-microglobulin (?2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ?2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ?2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ?2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ?2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ?2m amyloid-associated osteoarticular tissue destruction in DRA

Establishment of Protein Delivery Systems Targeting Podocytes

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes

Searching for new strategies against biofilm infections: Colistin-AMP combinations against Pseudomonas aeruginosa and Staphylococcus aureus single- and double-species biofilms

Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP) and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST) combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and tachyplesin I linear analogue (TP-I-L) was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) (http://www.fct.pt/), under the scope of the strategic funding of UID/B10/04469/2013 and COMPETE 2020 (POCI-01-0145-FEDER-006684). This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte. This work was also partially funded by the [14V105] Contract-Programme from the University of Vigo (https://mw.uvigo.gal/ uvigo_en/) and the Agrupamento INBIOMED (http://inbiomed.webs.uvigaes/) from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273) and co-financed by the European Regional Development Fund (http://ec.europleuiregionaL policy/EN/fundingierdf/) under the Operational Programme Innovative Economy (WNP-POIG.01.04.00-22-052/11).). Lipopharm.pl (http://www.lipopharm.p1/) provided support in the form of salaries for authors DG and WK. The authors also acknowledge the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (https://www.escmid.org/) for the Research Grant 2014 to Anglia Lourenco, and FCT for the PhD Grant of Paula Jorge (grant number SFRH/BD/88192/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

All-d-Enantiomer of β-Amyloid Peptide Forms Ion Channels in Lipid Bilayers

Alzheimer’s disease (AD) is the most common type of senile dementia in aging populations. Amyloid β (Aβ)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aβ-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aβ binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aβ peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aβ effects even in the absence of receptor–peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aβ isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aβ isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aβ isomer channels, and the d-isomer channel conductance is blocked by Zn2+, a known blocker of l-Aβ isomer channels. MD simulations further verify formation of β-barrel-like Aβ channels with d- and l-isomers, illustrating that both d- and l-Aβ barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca2+ leaking, unregulated channels in AD, and suggest that Aβ toxicity is mediated through a receptor-independent, nonstereoselective mechanism

Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

BACKGROUND: A number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2. CONCLUSIONS/SIGNIFICANCE: The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases