Periodic Reporting for period 3 - SynProAtCell (Delivery and On-Demand Activation of Chemically Synthesized and Uniquely Modified Proteins in Living Cells)
Berichtszeitraum: 2022-10-01 bis 2024-03-31
Despite great advances in protein research, the functions of many proteins remain insufficiently characterized. An important strategy used to probe some of the protein function is observing the effect of natural (posttranslational modifications, PTMs) and unnatural modifications on the protein activity. Protein modifications can be achieved by in vivo molecular biology approaches, however, even the best approaches are severely limited in their ability to introduce PTMs and unnatural modifications, including those that would allow for temporal control of protein function. At the same time, in vitro chemical protein synthesis allows for unlimited protein design and modification, but studies on delivering these synthetic proteins into cells are rare and are still limited to simple systems lacking atomic complexity, such as PTMs and temporal control elements. To answer fundamental questions about the cellular role of many proteins, and to enable monitoring protein’s cellular function and fate in real time, the fields of (semi)synthetic protein design and cellular protein manipulation must be bridged by significant advances in methods for protein delivery and real-time activation and monitoring.
In this ERC advanced project, we aim to develop a general approach for enabling considerably more detailed in-cell study of uniquely modified proteins by preparing proteins having the following features: 1) traceless cell delivery unit(s), 2) an activation unit for on-demand activation of protein function in the cell, and 3) a fluorescence probe for monitoring the state and the fate of the protein.
We will adopt this approach to shed light on the processes of ubiquitination and deubiquitination, which are critical cellular signals for many biological processes. We are currently employing our approach to study 1) the effect of inhibition of deubiquitinases in cancer. 2) Examining effect of phosphorylation on proteasomal degradation and on ubiquitin chain elongation. 3) Examining effect of covalent attachment of a known ligase ligand to a target protein on its degradation. Moreover, this could trigger the development of new methods to modify the desired protein in cell by selective chemistries and so rationally promote their degradation.
In this ERC advanced project, we aim to develop a general approach for enabling considerably more detailed in-cell study of uniquely modified proteins by preparing proteins having the following features: 1) traceless cell delivery unit(s), 2) an activation unit for on-demand activation of protein function in the cell, and 3) a fluorescence probe for monitoring the state and the fate of the protein.
We will adopt this approach to shed light on the processes of ubiquitination and deubiquitination, which are critical cellular signals for many biological processes. We are currently employing our approach to study 1) the effect of inhibition of deubiquitinases in cancer. 2) Examining effect of phosphorylation on proteasomal degradation and on ubiquitin chain elongation. 3) Examining effect of covalent attachment of a known ligase ligand to a target protein on its degradation. Moreover, this could trigger the development of new methods to modify the desired protein in cell by selective chemistries and so rationally promote their degradation.
The synthesis of the proposed proteins required further expansion of our toolbox for synthesizing challenging proteins. The following description highlights what we have achieved so far.
- Development of new solubilizing tag for hydrophobic proteins, which opened new horizons for studying Ub Hybrid Chains that are difficult to handle. This tag helped in the assembly of the peptide fragments and the HPLC purification steps along the protein synthesis.
- Development of highly efficient synthetic strategy for of disulfide bond containing peptides and proteins. Some of these targets are already marketed drugs.
- Development of new synthetic methods assisted by palladium complexes which will enable us to efficiently prepare uniquely synthetic proteins and activity-based probes.
The synthesis of key proteins in the project
- We synthesized the unmodified, mono- and doubly phosphorylated p19INK4d. We have shown the effect of phosphorylation on protein structure and stability.
- We synthesized homogenous ubiquitinated proteins bearing reporting tags; Flag, Myc and HA tags, for comparing 20S and 26S proteasomes with respect to substrate selection and peptide-product generation
Enhancing cellular uptake of synthetic proteins
- We introduced the DABCYL small molecule fused to cell penetrating peptide attached synthetic proteins (e.g. Ub) and observed threefold higher uptake efficacy in live cells compared to unmodified ones.
- We synthesized phosphatase stable ubiquitin probe and demonstrated its applicability to study how Ub phosphorylation affects its localization and conjugation by enzymatic machinery in human cells with minimal genetic manipulation.
- We developed a new technique for cell delivery, named multiplexed bead loading (MBL), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We have shown unprecedented involvements of Ub and SUMO2 in cellular processes depending on their conjugation states.
- Development of new solubilizing tag for hydrophobic proteins, which opened new horizons for studying Ub Hybrid Chains that are difficult to handle. This tag helped in the assembly of the peptide fragments and the HPLC purification steps along the protein synthesis.
- Development of highly efficient synthetic strategy for of disulfide bond containing peptides and proteins. Some of these targets are already marketed drugs.
- Development of new synthetic methods assisted by palladium complexes which will enable us to efficiently prepare uniquely synthetic proteins and activity-based probes.
The synthesis of key proteins in the project
- We synthesized the unmodified, mono- and doubly phosphorylated p19INK4d. We have shown the effect of phosphorylation on protein structure and stability.
- We synthesized homogenous ubiquitinated proteins bearing reporting tags; Flag, Myc and HA tags, for comparing 20S and 26S proteasomes with respect to substrate selection and peptide-product generation
Enhancing cellular uptake of synthetic proteins
- We introduced the DABCYL small molecule fused to cell penetrating peptide attached synthetic proteins (e.g. Ub) and observed threefold higher uptake efficacy in live cells compared to unmodified ones.
- We synthesized phosphatase stable ubiquitin probe and demonstrated its applicability to study how Ub phosphorylation affects its localization and conjugation by enzymatic machinery in human cells with minimal genetic manipulation.
- We developed a new technique for cell delivery, named multiplexed bead loading (MBL), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We have shown unprecedented involvements of Ub and SUMO2 in cellular processes depending on their conjugation states.
Recently we found non-proteinogenic cyclic peptides that binds Ub chains and capable to modulate its function. These peptides are rich in non-canonical features like N-methylation, which can tightly bind Lys48-linked Ub chains. The cyclic peptide appears to bind simultaneously to three Lys48-linked Ub units, block the action of protein degradation and induce apoptosis in vitro, and attenuate tumor growth in vivo.
To further increase the repertoire of the methods to preparer novel cyclic peptides that target the ubiquitin chain, we reported of a new method for rapid and efficient cyclization of uniquely modified peptides using gold complexes. The method features a fast reaction rate (within 20 min), mild conditions, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains. We are building on this approach to make new analogues of these cyclic peptides.
Expanding on using of gold complexes, we also reported a second new approach of generating cyclic peptides. We described a novel and efficient strategy of cyclization of unprotected peptides through rapid amine addition on a propagyl group to generate an imine linkage The applicability of this method was again demonstrated by the development of the cyclic peptide modulator for Lys48-linked di-Ub chains. The introduction of unnatural elements allowed us to overcome some of the drawbacks associated with peptide therapeutics, changing the nature of the linkage using the imine cyclization method led to a threefold increase in the apoptosis of these cyclic peptides in comparison with the ones reported earlier.
Alongside the finding and development of our first- and second-generation cyclic peptides we also developed a novel high-throughput strategy for screening ligands for Lys48-linked ubiquitin chains. Thanks to this approach we engineered dimeric peptides with an improved affinity.
The methods described above should enable us to better understand the cellular roles of proteins in particular to disease states. Such understanding will lead us to identify new targets in drug development. The Cyclic peptide we developed that modulate the Ub chains, with further development should lead to new therapeutic in cancer and open new horizon in validating such system in cancer in general. Moreover, the chemistry that was developed should find other uses in unrelated systems yet, still important to human health.
We plan to continue with our synthesis of the target proteins and achieve new advancement on their cellular activation and/or modulation with new chemical entities. We plan to the use the new delivery methods to test new chemistries in cells. For example, we are currently using the Spy-Catcher system to try to achieve protein degradation meditated by the Ub system. We plan to study how SUMO protein mediates PML formation, via its chemical synthesis in the caged form and then activate it in cell to measure the process in temporal and spatial resolution.
To further increase the repertoire of the methods to preparer novel cyclic peptides that target the ubiquitin chain, we reported of a new method for rapid and efficient cyclization of uniquely modified peptides using gold complexes. The method features a fast reaction rate (within 20 min), mild conditions, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains. We are building on this approach to make new analogues of these cyclic peptides.
Expanding on using of gold complexes, we also reported a second new approach of generating cyclic peptides. We described a novel and efficient strategy of cyclization of unprotected peptides through rapid amine addition on a propagyl group to generate an imine linkage The applicability of this method was again demonstrated by the development of the cyclic peptide modulator for Lys48-linked di-Ub chains. The introduction of unnatural elements allowed us to overcome some of the drawbacks associated with peptide therapeutics, changing the nature of the linkage using the imine cyclization method led to a threefold increase in the apoptosis of these cyclic peptides in comparison with the ones reported earlier.
Alongside the finding and development of our first- and second-generation cyclic peptides we also developed a novel high-throughput strategy for screening ligands for Lys48-linked ubiquitin chains. Thanks to this approach we engineered dimeric peptides with an improved affinity.
The methods described above should enable us to better understand the cellular roles of proteins in particular to disease states. Such understanding will lead us to identify new targets in drug development. The Cyclic peptide we developed that modulate the Ub chains, with further development should lead to new therapeutic in cancer and open new horizon in validating such system in cancer in general. Moreover, the chemistry that was developed should find other uses in unrelated systems yet, still important to human health.
We plan to continue with our synthesis of the target proteins and achieve new advancement on their cellular activation and/or modulation with new chemical entities. We plan to the use the new delivery methods to test new chemistries in cells. For example, we are currently using the Spy-Catcher system to try to achieve protein degradation meditated by the Ub system. We plan to study how SUMO protein mediates PML formation, via its chemical synthesis in the caged form and then activate it in cell to measure the process in temporal and spatial resolution.