Periodic Reporting for period 4 - HighResCells (A synergistic approach toward understanding receptor signaling in the cell at very high resolution)
Reporting period: 2023-09-01 to 2024-11-30
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 has been 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 has been 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.
This study focused on pivotal cell growth receptors, essential in both normal function and cancer progression. Since existing scientific tools were inadequate, new methodologies were developed, combining expertise across disciplines, including cryo-EM, computational analysis, and proteomics.
Two receptor classes were studied:
1. Epidermal Growth Factor Receptors (EGFRs) – Key regulators of cell growth, frequently implicated in cancer.
2. c-Met (Hepatocyte Growth Factor Receptor) – Linked to aggressive tumor behavior and poor prognosis.
A major breakthrough involved imaging HER2, a receptor overexpressed in breast cancer. Cryo-EM revealed a previously unknown conformation of HER2 bound to trastuzumab (Herceptin), interfering with HER2-HER3 complex formation. This discovery provides new insights into HER2-targeted therapy.
HER2 was further studied in cancer-derived membrane vesicles to visualize its natural interactions. For c-Met, expression patterns in different cancer cell lines were analyzed, with preliminary tomography studies conducted to explore its structural arrangement. Molecular markers were also developed to aid receptor identification in imaging experiments.
Importantly, mass spectrometry-based proteomics mapped EGFR signaling networks, identifying thousands of phosphorylation sites that regulate receptor activity. A key regulatory protein influencing EGFR degradation or recycling was uncovered, offering a potential therapeutic target.
These advances were made possible through interdisciplinary collaboration, combining imaging, molecular biology, and protein engineering expertise. Newly developed methodologies enhanced the study of receptor mechanisms, contributing to cancer research and drug development.
Two receptor classes were studied:
1. Epidermal Growth Factor Receptors (EGFRs) – Key regulators of cell growth, frequently implicated in cancer.
2. c-Met (Hepatocyte Growth Factor Receptor) – Linked to aggressive tumor behavior and poor prognosis.
A major breakthrough involved imaging HER2, a receptor overexpressed in breast cancer. Cryo-EM revealed a previously unknown conformation of HER2 bound to trastuzumab (Herceptin), interfering with HER2-HER3 complex formation. This discovery provides new insights into HER2-targeted therapy.
HER2 was further studied in cancer-derived membrane vesicles to visualize its natural interactions. For c-Met, expression patterns in different cancer cell lines were analyzed, with preliminary tomography studies conducted to explore its structural arrangement. Molecular markers were also developed to aid receptor identification in imaging experiments.
Importantly, mass spectrometry-based proteomics mapped EGFR signaling networks, identifying thousands of phosphorylation sites that regulate receptor activity. A key regulatory protein influencing EGFR degradation or recycling was uncovered, offering a potential therapeutic target.
These advances were made possible through interdisciplinary collaboration, combining imaging, molecular biology, and protein engineering expertise. Newly developed methodologies enhanced the study of receptor mechanisms, contributing to cancer research and drug development.
This project introduced innovative scientific methods to analyze cellular components with unprecedented detail, leveraging collaborative expertise to develop new imaging and computational tools.
New image processing techniques were devised to analyze small, flexible protein complexes, including Zernike-3D and SIREN-based approaches. These methods significantly improved structural reconstruction capabilities in biological studies.
To enhance imaging, novel vesicle production techniques were tested. Comparisons between naturally derived extracellular vesicles and those generated via extrusion allowed researchers to create optimal vesicles for studying membrane-bound proteins. Further refinements in vesicle purification, using engineered cleavable ligand columns, enabled selective enrichment of specific biological molecules.
To improve molecular tracking, engineered binding proteins (DARPins) were incorporated into icosahedral structures and DNA origami, allowing for precise labeling in cryo-EM and cryo-ET experiments. These markers provide a new approach to visualizing key biological structures.
In proteomics, a highly sensitive method—narrow-window data-independent acquisition (nDIA)—was optimized for advanced mass spectrometry. This method enhances protein identification and quantification in complex samples. Additionally, a streamlined protocol was developed for enriching extracellular vesicles from minimal biological samples, improving the study of vesicle-associated proteins.
These advancements have significantly refined imaging, molecular labeling, and protein analysis techniques, allowing for a more detailed understanding of biological processes. The integration of these innovations paves the way for future biomedical discoveries and therapeutic developments.
New image processing techniques were devised to analyze small, flexible protein complexes, including Zernike-3D and SIREN-based approaches. These methods significantly improved structural reconstruction capabilities in biological studies.
To enhance imaging, novel vesicle production techniques were tested. Comparisons between naturally derived extracellular vesicles and those generated via extrusion allowed researchers to create optimal vesicles for studying membrane-bound proteins. Further refinements in vesicle purification, using engineered cleavable ligand columns, enabled selective enrichment of specific biological molecules.
To improve molecular tracking, engineered binding proteins (DARPins) were incorporated into icosahedral structures and DNA origami, allowing for precise labeling in cryo-EM and cryo-ET experiments. These markers provide a new approach to visualizing key biological structures.
In proteomics, a highly sensitive method—narrow-window data-independent acquisition (nDIA)—was optimized for advanced mass spectrometry. This method enhances protein identification and quantification in complex samples. Additionally, a streamlined protocol was developed for enriching extracellular vesicles from minimal biological samples, improving the study of vesicle-associated proteins.
These advancements have significantly refined imaging, molecular labeling, and protein analysis techniques, allowing for a more detailed understanding of biological processes. The integration of these innovations paves the way for future biomedical discoveries and therapeutic developments.