Periodic Reporting for period 3 - HighResCells (A synergistic approach toward understanding receptor signaling in the cell at very high resolution)
Berichtszeitraum: 2022-03-01 bis 2023-08-31
The importance of this work for society lies in the fact that an understanding of the structure and dynamics of these receptors in their natural environment holds the key to develop better drugs, most importantly in the field of anti-cancer drugs. Only if we understand how the natural receptors act and interact can we design optimal molecules to prevent this with high specificity and thus few side effects for the patient. Therefore, the present work will have fundamental consequences for developing new anti-cancer medicines.
The overall objectives are to use defined membrane vesicles and whole cells, and employ 3D structure analysis by cryo-electron microscopy, especially tomography, greatly enhanced by novel image processing approaches, mass spectroscopy definitions of receptor modifications and interaction partners, as well as advanced protein engineering to identify, orient and freeze receptors for this method development. This collaborative project addresses the properties of such important receptors across a wide range of complexity and dimensions, in the cellular environment, through their high-resolution structures and changes during receptor recycling.
Moreover, the technology that will be developed will be generally applicable and may thus help to contribute to a paradigm change for structural biology, enabling atomic resolution description of receptors in their cellular environment.
As described above, the ultimate goal is to obtain near-atomic structures of membrane-bound receptors in the context of a cell. To achieve this goal, we exemplarily describe our approach towards HER2, which when overexpressed is one of the key drivers of breast and ovarian cancer. A purification strategy has been worked out to determine the single-particle structure of this receptor as a full-length protein, to obtain a reference material. This work has profited from generated binding proteins (DARPins) that permit the affinity-purification and in-situ labeling of the receptor.
Using HER2-overexpressing cancer cell lines, and a series of methods to create vesicles from these cells, cryo-electron tomography was used to obtain images of the receptor in the vesicle context. An important control was the proteomics analysis to identify the composition of the different vesicles obtained, and to follow the enrichment process. Conversely, the developed affinity reagents have permitted to enrich the vesicles that are particularly high in HER2.
A key component of this work is the 3D reconstruction of the receptor from the tomograms. Several methods were combined, ranging from the identification of segments of the membrane, classification in protein classes of the required size, their initial averaging, template-based methods and a series of newly developed mathematical tools. In an iteration between new methods of vesicle formation, proteomic identification, tomogram acquisition, image and model reconstruction, the work is approaching higher resolution of these receptors in the natural membrane context in a regular way.
Up to now, a massive technology development involving all 4 groups has taken place, involving protein expression, purification and engineering, analysis using advanced proteomics that is also focusing on the interaction partners within the cell, preparation and purification methods of vesicles, cryo-electron tomography and very many facets of image processing and model reconstruction.
We are confident that the project can be progressed to the point of identifying each single receptor molecule and obtaining very high-resolution 3D reconstructions until the end of the project. This will also include complexes with therapeutic antibodies, whose mode of action, therefore, might become visible for the first time, greatly helping in the understanding of the system. This, in turn, will be the basis for the design of improved therapeutic proteins with more advanced mode of action and possibly fewer side effects.