Periodic Reporting for period 4 - BENDER (BiogENesis and Degradation of Endoplasmic Reticulum proteins)
Período documentado: 2021-10-01 hasta 2022-06-30
Eukaryotic cells have lipid-enclosed microenvironments, ‘organelles’, that provide suitable environments for specific biochemical reactions. The Endoplasmic Reticulum (ER) is the site, where a major portion of protein synthesis occurs. Approximately one third of the human genome is part of the secretory pathway: they are synthesized at the ER membrane, mature further in the ER and are transported to their eventual destination, such as the exterior of the cell, the plasma membrane or intracellular organelles. The ER is equipped with an intricate protein network to govern the biogenesis and homeostasis of this large and diverse group of ‘secretory pathway’ proteins. The molecular mechanisms of how this machinery facilitates protein biogenesis and degradation are largely unknown to date. In this project, we use an integrative approach with cryo-electron tomography in its core to study the BiogENesis and Degradation of ER proteins (BENDER).
The overall goal of the BENDER project is to study the molecular architecture of the ER biogenesis and degradation machinery ‘in action’: 1) We focus on the static structure of the entry to the ER and secretory pathway, the ER translocon complex. This ‘swiss army knife’ can team up with accessory modules that meet the requirements of its various substrates and the state of the cell, 2) We study the maturation of proteins within the ER and 3) focus on how the ER maintains protein homeostasis under conditions of stress. To achieve these goals, we develop advanced computational methods for cryo-electron tomography that allow to most effectively distinguish different types of molecules involved in protein biogenesis and degradation as well as their different conformational states.
At the conclusion of this project, we have identified multiple intermediate states in the ER translation/translocation pathway, involving several new stoichiometric and substoichiometric components of the translocon. We have solved the structure of the human signal peptidase complex and gained significant new insight the mode of action of this complex that is essential for the maturation of proteins in the ER. We have characterized macromolecular changes that occur in the ER in response to protein-folding stress. In the process we have developed methods to localize specific macromolecules in cellular volumes and statistical analysis of their interactions, revealing protein biogenesis in the larger physiological context.
The overall goal of the BENDER project is to study the molecular architecture of the ER biogenesis and degradation machinery ‘in action’: 1) We focus on the static structure of the entry to the ER and secretory pathway, the ER translocon complex. This ‘swiss army knife’ can team up with accessory modules that meet the requirements of its various substrates and the state of the cell, 2) We study the maturation of proteins within the ER and 3) focus on how the ER maintains protein homeostasis under conditions of stress. To achieve these goals, we develop advanced computational methods for cryo-electron tomography that allow to most effectively distinguish different types of molecules involved in protein biogenesis and degradation as well as their different conformational states.
At the conclusion of this project, we have identified multiple intermediate states in the ER translation/translocation pathway, involving several new stoichiometric and substoichiometric components of the translocon. We have solved the structure of the human signal peptidase complex and gained significant new insight the mode of action of this complex that is essential for the maturation of proteins in the ER. We have characterized macromolecular changes that occur in the ER in response to protein-folding stress. In the process we have developed methods to localize specific macromolecules in cellular volumes and statistical analysis of their interactions, revealing protein biogenesis in the larger physiological context.
To study the biogenesis of specific proteins in a native-like environment using cryo-ET we have established a powerful ‘cell-free’ system. Membrane compartments extracted from cell lines (‘microsomes’) are biochemically functional in the sense that they facilitate the main post-translational modifications efficiently (i.e. signal peptide cleavage and glycosylation). We have extensively used this system for high-resolution imaging using cryo-electron tomography and have been able to identify 10 ribosomal intermediate states (4-8 Å resolution), 4 distinct ribosome-bound ER translocons, and 3 binding partners of the OST complex. Imaging in a physiological environment allowed us to determine how different types of translocons associate with polyribosomes that synthesize soluble or multipass-transmembrane proteins, respectively. The use of cell culture also allowed us to determine the reorganization of ER-associated ribosomes and the translocon to ER stress.
To investigate the further maturation of proteins we have determined the structure of the isolated human signal peptidase complex (SPC). We showed that the human SPC consists of two paralogs and the structure suggests that the specificity SPC for signal peptide is achieved through thinning the ER membrane locally, excluding off-target proteins.
To study the ER in situ, we set up workflow that combines cryo-fluorescence light microscopy with cryo-focused ion beam milling and cryo-electron tomography. This workflow was used to characterize ER-related macromolecular changes of both short- and long-term protein-folding stress (UPR) in yeast. This work is still ongoing, but we have already observed several UPR-induced changes to the ER that could help reduce ER protein load and increase ER-folding capacity.
Key for high-resolution structural studies using tomography are our developments in tomography processing. To localize and distinguish different types of molecules in the cell we developed a ‘deep-learning’ based approach. We have established a community-wide contest to assess the progress of methods for localization and identification of macromolecules in tomograms and monitor the progress in the field. All software innovations achieved during BENDER are part of our software package pytom (www.pytom.org).
Altogether, the work has resulted in 14 scientific publications at the closure of BENDER, with several more in progress.
To investigate the further maturation of proteins we have determined the structure of the isolated human signal peptidase complex (SPC). We showed that the human SPC consists of two paralogs and the structure suggests that the specificity SPC for signal peptide is achieved through thinning the ER membrane locally, excluding off-target proteins.
To study the ER in situ, we set up workflow that combines cryo-fluorescence light microscopy with cryo-focused ion beam milling and cryo-electron tomography. This workflow was used to characterize ER-related macromolecular changes of both short- and long-term protein-folding stress (UPR) in yeast. This work is still ongoing, but we have already observed several UPR-induced changes to the ER that could help reduce ER protein load and increase ER-folding capacity.
Key for high-resolution structural studies using tomography are our developments in tomography processing. To localize and distinguish different types of molecules in the cell we developed a ‘deep-learning’ based approach. We have established a community-wide contest to assess the progress of methods for localization and identification of macromolecules in tomograms and monitor the progress in the field. All software innovations achieved during BENDER are part of our software package pytom (www.pytom.org).
Altogether, the work has resulted in 14 scientific publications at the closure of BENDER, with several more in progress.
Structural studies of specific processes in the cell are essential to understand their underlying chemistry and to possibly alter these processes. Traditionally, such structure-function studies are carried out using isolated molecules and macromolecular complexes. However, progress in our structural and mechanistic understanding of processes at membranes mediated by transient interactions is slow because those are difficult to reconstitute biochemically. The difficulties lie in the many components involved and in the lipid environment – traditional approaches require solubilization, which fundamentally changes the biochemical environment of the molecules. Combining cutting-edge cryo-electron tomography with advances in computational volume analysis have provided a unique opportunity for analyzing these processes ‘top down’ rather than ‘bottom up’, avoiding the limitations of traditional reconstitution-based approaches. Due to its high cellular abundance the ER-associated protein synthesis and degradation machinery was a natural starting point for this emerging approach to structural biology. Using this approach, we could determine a near-complete atomic model of the protein synthesis machine (ribosome) together with a complex consisting of the protein translocation channel Sec61, the enzyme responsible for N-glycosylation (Oligosaccharyltransferase), and the translocon-associated protein complex. Our in-depth analysis of the ER translocon has revealed further, less abundant ER translocons and their interplay during translation. Unexpectedly, the analysis of ribosomes also revealed new insights into mRNA translation in mammalian cell. Key to these discoveries complementing studies of limited reconstituted systems was the analysis of protein translation and biogenesis in the context of polysomes in their native settings.
Despite its essential function in the cell and a Nobel prize awarded to Gunter Blobel, the structure of the signal peptidase complex (SPC) and its precise molecular mechanism ensuring its function with high specificity remained elusive over almost half a century. Our SPC structure has provided key insight into the substrate recognition mechanism of this complex. The novel concept emerging from our structure is that it achieves specificity through a thinned lipid microcompartment. The structure and molecular principles emerging from it could open the possibility of developing drugs to target specific microbial or viral SPs.
Despite its essential function in the cell and a Nobel prize awarded to Gunter Blobel, the structure of the signal peptidase complex (SPC) and its precise molecular mechanism ensuring its function with high specificity remained elusive over almost half a century. Our SPC structure has provided key insight into the substrate recognition mechanism of this complex. The novel concept emerging from our structure is that it achieves specificity through a thinned lipid microcompartment. The structure and molecular principles emerging from it could open the possibility of developing drugs to target specific microbial or viral SPs.