Periodic Reporting for period 1 - MicroProtLip (New microfluidic tools for characterization of protein-lipid interactions)
Berichtszeitraum: 2020-07-01 bis 2022-06-30
Cellular function and malfunction depend crucially on the interactions of proteins and other cellular components. Interactions specifically with lipid membranes are key to the compartmentalization of a wide range of cellular mechanisms into discrete organelles, allowing to maintain homeostasis. A detailed understanding of the mechanisms by which lipid surfaces take part in the control of protein function is therefore of fundamental importance. Understanding these interactions also has important pharmaceutical implications, as ~60% of drug targets are known to be membrane-associated proteins. Despite the implications to life sciences and drug discovery, our current understanding of protein–lipid interactions is limited. This is primarily due to the challenges of existing methods to accurately determine the interactions between proteins and lipid bilayers due to the high level of heterogeneity of the formed complexes.
Recent advances in the development of microfluidic techniques that allow the characterisation of biomolecular complexes together with the availability of sensitive optical detection methods however, open new possibilities for fundamental studies of protein–lipid interactions. The project MicroProtLip focused on overcoming current limitations by establishing a novel, game-changing experimental microfluidic platform to elucidate the dynamic membrane protein interactions in a lipid bilayer context, and in a qualitative and quantitative manner. This project was performed at the University of Cambridge as well as Fluidic Analytics, a company that has pioneered the development of a microfluidic platform that allows the characterization of biomolecular interactions in solution and without surface constraints. The main objective of the MicroProtLip project was the development of transformative assays for microfluidic platforms dedicated for the study of interactions of lipid membranes with protein molecules. Microfluidic technologies, that allow for manipulation of liquids at the microscale, were used for dividing complex samples into fractions. By bringing together single-molecule interrogation with microfluidics, we are aiming to bridge the gap of a missing technology that enables probing interactions and self-assembly of proteins within a lipid bilayer context.
Our novel approach was used to address questions that concern a wide range of aspects of mechanism of protein–lipid interaction such as the mechanism by which α-synuclein – a protein linked genetically and neuropathologically to Parkinson’s disease – binds to lipid surfaces. Interplay between α-synuclein and lipid surfaces was reported to be important for both the physiological function of protein as well as to induce aggregation (a hallmark of the development of Parkinson’s Disease). The focus is on the ability of transient toxic oligomeric forms to bind to lipids and disrupt vesicles. We also utilized this new approach to detect proteins present on the surface of extracellular vesicles – lipid vesicles produced by cells and circulating in body fluids. Proteins displayed on the surface and inside of exosomes provide unique information about the molecular characteristics of the microenvironment and have vast potential in the early detection and monitoring of the progression of cancer. Characterization of the vesicles that are present at low concentrations with sensitive methods will advance the novel approach in diagnostics of early signs of disease. The exemplary problems addressed in the MicroProtLip project have wide implications for society, by adding towards a better understanding of molecular processes in increasingly more prevalent diseases that could in the future lead to the development of new drugs.
Recent advances in the development of microfluidic techniques that allow the characterisation of biomolecular complexes together with the availability of sensitive optical detection methods however, open new possibilities for fundamental studies of protein–lipid interactions. The project MicroProtLip focused on overcoming current limitations by establishing a novel, game-changing experimental microfluidic platform to elucidate the dynamic membrane protein interactions in a lipid bilayer context, and in a qualitative and quantitative manner. This project was performed at the University of Cambridge as well as Fluidic Analytics, a company that has pioneered the development of a microfluidic platform that allows the characterization of biomolecular interactions in solution and without surface constraints. The main objective of the MicroProtLip project was the development of transformative assays for microfluidic platforms dedicated for the study of interactions of lipid membranes with protein molecules. Microfluidic technologies, that allow for manipulation of liquids at the microscale, were used for dividing complex samples into fractions. By bringing together single-molecule interrogation with microfluidics, we are aiming to bridge the gap of a missing technology that enables probing interactions and self-assembly of proteins within a lipid bilayer context.
Our novel approach was used to address questions that concern a wide range of aspects of mechanism of protein–lipid interaction such as the mechanism by which α-synuclein – a protein linked genetically and neuropathologically to Parkinson’s disease – binds to lipid surfaces. Interplay between α-synuclein and lipid surfaces was reported to be important for both the physiological function of protein as well as to induce aggregation (a hallmark of the development of Parkinson’s Disease). The focus is on the ability of transient toxic oligomeric forms to bind to lipids and disrupt vesicles. We also utilized this new approach to detect proteins present on the surface of extracellular vesicles – lipid vesicles produced by cells and circulating in body fluids. Proteins displayed on the surface and inside of exosomes provide unique information about the molecular characteristics of the microenvironment and have vast potential in the early detection and monitoring of the progression of cancer. Characterization of the vesicles that are present at low concentrations with sensitive methods will advance the novel approach in diagnostics of early signs of disease. The exemplary problems addressed in the MicroProtLip project have wide implications for society, by adding towards a better understanding of molecular processes in increasingly more prevalent diseases that could in the future lead to the development of new drugs.
The MicroProtLip project resulted in the development of microfluidic technologies, that are based on electrophoretic separation of species. Therefore, they are suitable for characterization of complex samples, such as protein–lipid complexes. We have demonstrated that the proposed technologies can be successfully used for answering important questions in biology.
First, we explored the mechanism of α-synuclein oligomers binding to lipid membranes. Oligomers are low molecular weight transient assemblies of monomers, which are suspected to have toxic effects through aberrant interactions with cell membranes. We found that oligomers selectively bind to negatively charged lipid membranes, and the curvature of the membrane is important for oligomer-lipid vesicle interaction. Oligomers displayed much higher affinity towards lipid membranes than monomers and could displace monomers from the surface. Both oligomers and monomers reduced the effective charge of liposomes but form different types of assemblies with lipid vesicles. The results of this project were presented to scientific community during two conferences and the manuscript for publication in a peer-reviewed journal is in preparation.
We further characterized exosomes – small lipid vesicles, that are produced and released by all types of cells. We precisely quantified not only the number of vesicles, but also counted the number of cancer biomarker molecules on single exosomes. The results of the digital detection of cancer markers were recently accepted for publication in Nature Communications journal.
First, we explored the mechanism of α-synuclein oligomers binding to lipid membranes. Oligomers are low molecular weight transient assemblies of monomers, which are suspected to have toxic effects through aberrant interactions with cell membranes. We found that oligomers selectively bind to negatively charged lipid membranes, and the curvature of the membrane is important for oligomer-lipid vesicle interaction. Oligomers displayed much higher affinity towards lipid membranes than monomers and could displace monomers from the surface. Both oligomers and monomers reduced the effective charge of liposomes but form different types of assemblies with lipid vesicles. The results of this project were presented to scientific community during two conferences and the manuscript for publication in a peer-reviewed journal is in preparation.
We further characterized exosomes – small lipid vesicles, that are produced and released by all types of cells. We precisely quantified not only the number of vesicles, but also counted the number of cancer biomarker molecules on single exosomes. The results of the digital detection of cancer markers were recently accepted for publication in Nature Communications journal.
The technological progress achieved in the MiroProtLip project allows for further use of microfluidic platforms for the studies on the interactions of proteins with lipid vesicles. The experiments performed in the project address important questions associated with the development of diseases, that become more prevalent in society. In the pathology of Parkinson’s Disease, the activity of the oligomeric form of the protein α-synuclein was recently identified as potentially toxic to the integrity of lipid surfaces in cells. Through detailed characterization of oligomer–lipid interactions we expand the current knowledge on the mechanism of interactions, and introduce a platform, that will allow for quick assessment of the influence of, for example, potential drug molecules on the affinity of oligomers to the lipid membranes. We also show the capability of the microfluidic platform to be used for quantification of cancer biomarkers. Potentially, the method could be developed further into a diagnostic assay for monitoring the progression of disease and therapy from the blood derived patient samples.