Periodic Reporting for period 3 - HighResCells (A synergistic approach toward understanding receptor signaling in the cell at very high resolution)
Periodo di rendicontazione: 2022-03-01 al 2023-08-31
The key problem to be addressed in the current proposal is the study of some of the most pivotal growth receptors of the cell at high resolution in their cellular context. Members of the Epidermal Growth Factors Receptor family (EGFRs) influence cell growth and proliferation and are critical in all phases of tumor progression. Therefore, they have been the target of many therapeutic interventions, such as therapeutic antibodies, kinase inhibitors but also many novel experimental therapies aiming to inhibit their function to control tumor growth. Despite their enormous importance, the exact structure of these receptors within the natural cell membrane, their detailed interaction with other cellular factors and their exact interaction with therapeutic substances is not understood. The impact of having a precise structural understanding as well as their dynamics of interaction can hardly be overestimated. Moreover, the technical developments emerging out of this project serve as a blueprint to study other important cell receptors in an analogous manner.
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.
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.
The work performed is of a highly interdisciplinary nature and has combined protein engineering, cryo-electron microscopy, structural computation and proteomic analysis in an exemplary way. As will be clear from the following more detailed account, only this collaboration across disciplines could have made the progress in the project possible that has been achieved up to now.
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.
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.
Full length receptors have not been studied at high resolution, and much less so in the context of the natural biological membrane in the context of cell-derived vesicles. All of this is therefore beyond the state of the art.
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.
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.