Final Report Summary - AMYLOID (Membrane activity of amyloid fibrils: unravelling cell killing mechanism)
A broad spectrum of devastating pathological conditions, including type II diabetes, Alzheimer’s and Parkinson's diseases are classified under a common type of disorders known as amyloidosis. Disease onset is triggered by transformation of soluble proteins from their native three-dimensional structure into highly stable filamentous assemblies which progressively accumulate in cells and tissues. Remarkably, despite the finding that the proteins causing different amyloid disorders are completely unrelated to each other in terms of primary sequence, structure and function, the resulting aggregates have a common fibrillar structure which is characterized by a specific intermolecular alignment of -strands, termed a cross-β-structure. Such similarity in the structural arrangement of the harmful protein agents implies that different diseases may manifest from similar mechanisms of cytotoxicity. For many years, however, small soluble intermediates of the aggregation pathway, rather than mature amyloid deposits, have been regarded as the primary causative agents that lead to cell death. More recently, however, the role of amyloid fibrils in the disease has been reconsidered based on new findings which have implicated protein fibrils in cellular dysfunction. Furthermore, the extent of cellular damage was found to correlate with the ability of fibrillar species to destroy biomembranes. The mode of action by which fibrils mediate cell membrane disruption, however, is still largely unexplored. In his project we aimed to address this important question by investigating distinct aspects of fibril-membrane interactions. The study selected β2-microglobulin (β2m), a member of immunoglobulin fold of proteins structures, as a model system for studying amyloid-induced membrane damage. β2m is involved in the human disorder dialysis-related amyloidosis, a pathological condition occurring in more than 700,000 patients worldwide. The protein has been shown to form fibrillar deposits in joints of the affected individuals and also to assemble into amyloid fibrils in vitro.
Maintaining cell membrane integrity is a fundamental requirement for sustaining cells in a viable and healthy condition, since an intact lipid barrier is vital for almost all known physiological processes. Using fluorescence spectroscopy we observed previously that β2m fibrils disrupt model vesicles composed of negatively charged and zwitterionic lipids, making them permeable to water soluble small molecules. The results of the current study, obtained by fluorescence microscopy and cryo-electron microscopy and tomography, revealed that β2m fibrils disrupt membrane integrity by causing extensive lipid removal from the vesicles. Binding of the extracted lipids to the fibrils was evident through detection of a thin lipid layer covering the fibrillar aggregates. Cryo-electron tomograms recorded in collaboration with Prof. Helen Saibil (Birkbeck College, London) provided further evidence for membrane damage on a nanometer scale. These experiments allowed separate steps of membrane destruction mediated by amyloid fibrils to be visualized at high resolution for the first time. EM images revealed that fibril binding induces deformation of the vesicles, causing them to adopt a drop-shaped appearance. Furthermore, we observed blebbing and breakage of the outer membrane leaflet and de-novo formation of small vesicular structures, presumably assembled from the extracted lipids. Fragmented β2m fibrils, generated from their full-length counterparts by controlled mechanical stirring, induced a significantly greater extent of the membrane damage than the longer, parent fibrillar species. These results demonstrate that fibril fragmentation, a process which is likely to be occurring in vivo, amplifies the amount of membrane-active entities which appear to involve fibril ends.
One of the most important and demanding questions in the field of amyloidosis is how to sequester toxic protein aggregates from interactions with their targets. In this study we assessed two classes of bio-molecules for their ability to reduce membrane disruption by β2m fibrils: glycosaminoglycans (GAGs), anionic polysaccharides widely expressed in different tissue types; and polyphenols, aromatic substances exhibiting anti-oxidant and anti-inflammatory properties. GAG compounds included heparin and its building subunit heparin disaccharide, while the polyphenolic group of compounds included the naturally occurring epigallocatechin gallate (EGCG) (found in tea leaves), resveratrol (from red grapes) and a synthetic dye, bromophenol blue. These molecules were selected for this study because they are known to interact with, and modulate, fibril formation for several amyloidogenic proteins. Our study was designed to test whether these substances also prevent membrane distortion and damage by amyloid fibrils. Importantly, the morphology of the β2m fibrils was unaffected by the tested compounds during the course of the experiments. Heparin proved to be the most powerful substance in suppressing fibril-induced membrane damage among the compounds examined. This molecule was able to abolish vesicle deformation and lipid extraction as judged by confocal microscopy. Strikingly, despite having the same charge and basic structure as the parent polymer, heparin disaccharide was unable to inhibit membrane disruption. These results suggest that electrostatic interactions are important, but not sufficient, for preventing damage of lipid vesicles by β2m fibrils. Instead, multiple binding groups of full-length heparin are required to amplify its affinity for the fibrils in order to obtain the observed inhibitory effect. Amongst the three polyphenolic molecules studied, only EGCG was able to attenuate fibril-mediated membrane disruption, although this compound was less efficient than heparin. These results are not only critical for the design of future therapeutic strategies that may ameliorate amyloid disorders, but are equally important for development deeper understanding of principal forces that govern fibril-lipid association.
Different cellular organelles contain distinct lipid compositions and intra-luminal environments, such as pH. Membranes of late endosomes and lysosomes, the main components of the endocytic pathway, contain a unique lipid molecule termed bis(monoacylglycero)phosphate (BMP) and sustain an acidic pH. We hypothesised that an enhanced affinity of amyloid fibrils toward particular membrane lipids may render specific organelles as the cellular targets of amyloid-imposed damage and hence amyloid disease. To investigate this possibility, we examined membrane activity of β2m fibrils using lipids and pH values that mimic lysosomes, an organelle implicated in many amyloid disorders. These experiments showed that the fibrils caused extensive membrane disruption of BMP-containing vesicles, while lower pH increased this effect. Our findings suggest that BMP, having four unsaturated hydrocarbon chains, destabilises the lipid bilayer, especially in combination with an acidic pH. The study implies that the interactions of amyloid fibrils with lysosomal membranes could be one of the factors resulting in the previously reported dysfunction of this organelle in amyloid disorders.
Protein misfolding diseases affect millions of people worldwide, but despite enormous research efforts in this field, only supporting treatments are available. Our research work addressed a fundamental biological question with global biomedical and healthcare importance. The results obtained have advanced our understanding of the mechanism by which β2m amyloid fibrils give rise to membrane destruction and also suggested potential new routes for prevention of these harmful effects. All these are important steps towards the design of future therapeutic avenues. Further studies are required to assess whether our findings are relevant to other amyloidogenic proteins and hence whether there is a possibility to treat additional amyloid diseases by interruption of lipid-fibril interactions.
Maintaining cell membrane integrity is a fundamental requirement for sustaining cells in a viable and healthy condition, since an intact lipid barrier is vital for almost all known physiological processes. Using fluorescence spectroscopy we observed previously that β2m fibrils disrupt model vesicles composed of negatively charged and zwitterionic lipids, making them permeable to water soluble small molecules. The results of the current study, obtained by fluorescence microscopy and cryo-electron microscopy and tomography, revealed that β2m fibrils disrupt membrane integrity by causing extensive lipid removal from the vesicles. Binding of the extracted lipids to the fibrils was evident through detection of a thin lipid layer covering the fibrillar aggregates. Cryo-electron tomograms recorded in collaboration with Prof. Helen Saibil (Birkbeck College, London) provided further evidence for membrane damage on a nanometer scale. These experiments allowed separate steps of membrane destruction mediated by amyloid fibrils to be visualized at high resolution for the first time. EM images revealed that fibril binding induces deformation of the vesicles, causing them to adopt a drop-shaped appearance. Furthermore, we observed blebbing and breakage of the outer membrane leaflet and de-novo formation of small vesicular structures, presumably assembled from the extracted lipids. Fragmented β2m fibrils, generated from their full-length counterparts by controlled mechanical stirring, induced a significantly greater extent of the membrane damage than the longer, parent fibrillar species. These results demonstrate that fibril fragmentation, a process which is likely to be occurring in vivo, amplifies the amount of membrane-active entities which appear to involve fibril ends.
One of the most important and demanding questions in the field of amyloidosis is how to sequester toxic protein aggregates from interactions with their targets. In this study we assessed two classes of bio-molecules for their ability to reduce membrane disruption by β2m fibrils: glycosaminoglycans (GAGs), anionic polysaccharides widely expressed in different tissue types; and polyphenols, aromatic substances exhibiting anti-oxidant and anti-inflammatory properties. GAG compounds included heparin and its building subunit heparin disaccharide, while the polyphenolic group of compounds included the naturally occurring epigallocatechin gallate (EGCG) (found in tea leaves), resveratrol (from red grapes) and a synthetic dye, bromophenol blue. These molecules were selected for this study because they are known to interact with, and modulate, fibril formation for several amyloidogenic proteins. Our study was designed to test whether these substances also prevent membrane distortion and damage by amyloid fibrils. Importantly, the morphology of the β2m fibrils was unaffected by the tested compounds during the course of the experiments. Heparin proved to be the most powerful substance in suppressing fibril-induced membrane damage among the compounds examined. This molecule was able to abolish vesicle deformation and lipid extraction as judged by confocal microscopy. Strikingly, despite having the same charge and basic structure as the parent polymer, heparin disaccharide was unable to inhibit membrane disruption. These results suggest that electrostatic interactions are important, but not sufficient, for preventing damage of lipid vesicles by β2m fibrils. Instead, multiple binding groups of full-length heparin are required to amplify its affinity for the fibrils in order to obtain the observed inhibitory effect. Amongst the three polyphenolic molecules studied, only EGCG was able to attenuate fibril-mediated membrane disruption, although this compound was less efficient than heparin. These results are not only critical for the design of future therapeutic strategies that may ameliorate amyloid disorders, but are equally important for development deeper understanding of principal forces that govern fibril-lipid association.
Different cellular organelles contain distinct lipid compositions and intra-luminal environments, such as pH. Membranes of late endosomes and lysosomes, the main components of the endocytic pathway, contain a unique lipid molecule termed bis(monoacylglycero)phosphate (BMP) and sustain an acidic pH. We hypothesised that an enhanced affinity of amyloid fibrils toward particular membrane lipids may render specific organelles as the cellular targets of amyloid-imposed damage and hence amyloid disease. To investigate this possibility, we examined membrane activity of β2m fibrils using lipids and pH values that mimic lysosomes, an organelle implicated in many amyloid disorders. These experiments showed that the fibrils caused extensive membrane disruption of BMP-containing vesicles, while lower pH increased this effect. Our findings suggest that BMP, having four unsaturated hydrocarbon chains, destabilises the lipid bilayer, especially in combination with an acidic pH. The study implies that the interactions of amyloid fibrils with lysosomal membranes could be one of the factors resulting in the previously reported dysfunction of this organelle in amyloid disorders.
Protein misfolding diseases affect millions of people worldwide, but despite enormous research efforts in this field, only supporting treatments are available. Our research work addressed a fundamental biological question with global biomedical and healthcare importance. The results obtained have advanced our understanding of the mechanism by which β2m amyloid fibrils give rise to membrane destruction and also suggested potential new routes for prevention of these harmful effects. All these are important steps towards the design of future therapeutic avenues. Further studies are required to assess whether our findings are relevant to other amyloidogenic proteins and hence whether there is a possibility to treat additional amyloid diseases by interruption of lipid-fibril interactions.