The membrane as a catalyst of damaging protein misfolding events

The unit of life, the cell, is enclosed by the cell membrane, a thin barrier just a few nanometers thick. This is not a solid barrier, but consists of lipids and other biomolecules that together behave like a semi-liquid oil-like film. In this film, there are also delimited areas that can have different functions and properties. In this complex and ever-changing environment of the membrane, it is a challenge to understand how each component affects each other. This project investigates how lipids affect proteins and how proteins affect lipids and the membrane. One of the most important questions is whether any lipids or physical conditions in the membrane can cause certain proteins to misfold. Proteins, which are long chains of amino acids, normally need to twist into a specific structure, or fold, to function. If this (normally) spontaneous process is disturbed, they can instead form harmful structures. This is called protein misfolding. This project investigates whether some lipids can cause certain membrane-associated proteins to misfold (or possibly fold more slowly) than they normally would. Protein misfolding is involved in many diseases, including Parkinson's, Alzheimer's and Diabetes II, as well as prion diseases. Insight into the role of the lipid membrane can potentially and over time lead to better prevention and treatment of these disease. Our findings confirm that cells have tissue-specific lipid profiles and significantly modulate their lipid composition throughout their life cycle, and also that their lipid profiles change with aging. The lipid mixtures can be so different that the physical properties of the membrane of which they are a part change significantly, which in turn can affect protein folding. There are also significant changes in lipid molecules involved in how the cell communicates with itself. Misfolded proteins that are in contact with the membrane can also destroy its integrity when the cell is in a particularly vulnerable situation, or affect lipid-based signalling. A lipid molecule that to a certain extent correlates with the development of Parkinson's and Alzheimer's Diseases, is cholesterol. However, it is unclear whether this is a molecular cause or consequence of the disease state, or unrelated. An important finding in our studies shows that cholesterol accelerates the folding of a Parkinson's-related protein by a factor of 20 or more. Other membrane compositions are found to have an inhibitory effect. In sum, this project strengthens the hypothesis put forward by this project: that the membrane is an important and sensitive platform for molecular processes relevant to neurodegenerative diseases.

The project proved that lipids are both modulated as a function of cell type, cell cycle point and senescence, and results also suggest that these differences can play a role in protein function and dysfunction through folding/misfolding events. (Objective 1) Membrane can drive misfolding by forcing the protein to expose its hydrophobic interiors in new ways, and/or align the denatured polypeptides along the membrane surface. Lipids achieve this through charge-matching, headgroup mismatch and hydrogen bonding. (Objective 2) This was found to be true: cholesterol and liquid-ordered states promote misfolding and the formation of harmful aggregates for alpha-synulcein. (Objective 3) Amphitropic proteins tend to be attracted to lipid charge, and singalling phosphoinesotides will be targeted. (Objective 4) The correct balance between protein, membrane charge and order/disorder is important, too much cholesterol is a promoter of adverse molecular outcomes.

Proteins and the cell membrane are functionally intimately connected. Membrane proteins must fold and embed into the membrane, and proteins associating with the membrane must not be excessively perturbed by the membrane, or affect its integrity. Given the flexible structure of many of these membrane-associating proteins, their high effective concentrations at the membrane and the attenuation of energetic barriers between different folding states, many may be at high risk for misfolding. Annular Oligomers (AOs) are an example of such a misfolding event. AOs are considered highly toxic and are implicated in neurodegenerative and prionic diseases. Their formation (into pore-like aggregates of 30-150 nm) is prompted by still poorly understood mechanisms that often occur in conjunction with the lipid membrane. Little is known about the mechanism of misfolding, or how this is relevant in living cells. This project seeks to elucidate these issues by examining AO-forming peptides and a protein-fatty acid (PFA)-complex which interacts with the membrane and forms AOs. The project proposes that lipid- and lipid-like factors such as fatty acids catalyze the formation of AOs. It aims to find specific protein-lipid interactions and transition states that underpin such a mechanism. It also proposes that differences in the lipid composition and lateral organization of membranes will make some cells more or less susceptible to AO action. AFM is employed as the main techniques to identify and explore AOs and how they form in artificial lipid systems. Then, the project combines high-resolution NMR with molecular dynamics to provide the details necessary to investigate transition-states between soluble, membrane bound, and AO-forming situations. LC-MS/MS and analytical NMR is combined with phase-sensitive fluorescence studies to provide a detailed picture of membranes (cell and artificial) that promote or suppress AO-formation.