Periodic Reporting for period 1 - RiboFriend (Deciphering co-translational protein folding, assembly and quality control pathways, in health and disease)
Período documentado: 2022-04-01 hasta 2024-09-30
The majority of cellular proteins do not function alone; rather acting together to achieve concerted functions. Despite the prevalence of protein complexes, little is known of the mechanisms that ensure their correct folding and assembly in the crowded cytoplasm. The importance of the folding challenge is underscored by the growing number of misfolding diseases, often characterized by aggregation of lonely, unassembled protein-subunits.
At the critical intersection of synthesis and folding, the ribosome is emerging as a hub, guiding the polypeptide-chain interactions with targeting factors, modifying enzymes and folding chaperones. We have recently discovered that even the final step of folding, the assembly into complexes, is coordinated with translation. To capture co-translational events, in vivo, we developed a ribosome profiling approach. This approach revealed that emerging polypeptide-chains are constantly engaged by their partner subunits, protecting them from misfolding. However the mechanisms regulating co-translational assembly pathways remain largely obscure. In this proposal, we aim to elucidate co-translational protein folding and degradation mechanisms, in health and disease.
We will: (i) Identify and characterize novel co-translational degradation pathways, by targeting ribosomes synthesizing misfolding-prone subunits. (ii) Elucidate the conformational, energetic and kinetic parameters directing folding and assembly, at the atomic level, by molecular dynamics (iii) Develop RiboFriend a single-molecule in vivo approach to elucidate the interplay of chaperones, degradation and assembly factors, during synthesis. Our collective preliminary results strongly support the feasibility of this proposal. The ability to capture co-translational folding and misfolding pathways, in single-molecule resolution can revolutionize our understanding of conformational diseases and the aging process, opening new horizons for therapy.
At the critical intersection of synthesis and folding, the ribosome is emerging as a hub, guiding the polypeptide-chain interactions with targeting factors, modifying enzymes and folding chaperones. We have recently discovered that even the final step of folding, the assembly into complexes, is coordinated with translation. To capture co-translational events, in vivo, we developed a ribosome profiling approach. This approach revealed that emerging polypeptide-chains are constantly engaged by their partner subunits, protecting them from misfolding. However the mechanisms regulating co-translational assembly pathways remain largely obscure. In this proposal, we aim to elucidate co-translational protein folding and degradation mechanisms, in health and disease.
We will: (i) Identify and characterize novel co-translational degradation pathways, by targeting ribosomes synthesizing misfolding-prone subunits. (ii) Elucidate the conformational, energetic and kinetic parameters directing folding and assembly, at the atomic level, by molecular dynamics (iii) Develop RiboFriend a single-molecule in vivo approach to elucidate the interplay of chaperones, degradation and assembly factors, during synthesis. Our collective preliminary results strongly support the feasibility of this proposal. The ability to capture co-translational folding and misfolding pathways, in single-molecule resolution can revolutionize our understanding of conformational diseases and the aging process, opening new horizons for therapy.
To address aim (i), we targeted ribosomes translating misfolding-prone proteins. Utilizing a combination of proteomics and ribosome profiling approaches, we were able to develop a highly sensitive proteomics approach to identify co-translational interactors. These specific interactors include known factors involved in the ubiquitin-proteasome pathway, as well as several putative E3 ligases and folding chaperones. Importantly, many of the discovered factors are entirely novel. Based on this, we compiled a candidate list of ribosome quality-control factors for further analysis. All candidates’ deletion strains displayed impaired growth phenotype when exposed to various translation stresses. Several candidates were also found to be required for co-translational degradation of several misfolding-prone proteins. In order to reveal the impact on nascent-chains proteolysis in a proteome-wide manner, we developed a method to map N`-terminal peptides integrity. We are combining this with selective ribosome profiling of the candidates.
To address aim (ii), we combined selective ribosome profiling, imaging, and advanced proteomics with all-atoms molecular dynamics. Focusing on the conserved N-terminal acetyltransferases (NATs) family, we revealed diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We demonstrated that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harbouring hotspots exhibited high levels of thermo-lability, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complex assembly. We next demonstrated that this assembly disruption led to subunits aggregation, analysed by advanced imaging approaches. Accordingly, conservation analysis revealed that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include more complexes, we find similar trends, allowing us to propose a predictive model.
To address aim (iii), we developed a single-molecule ribosome profiling approach, termed RiboFriend. We relied on our experience in combining biochemistry and sequencing to develop a nanopore based approach, to capture the interplay of factors guiding co-translational folding, in vivo. We have generated two chimeric constructs, fusing each co-translationally acting factor with a different RNA modifying enzyme. Direct RNA sequencing using NanoPore revealed that over 700 RNA transcripts exhibit significant mRNA modifications.
To address aim (ii), we combined selective ribosome profiling, imaging, and advanced proteomics with all-atoms molecular dynamics. Focusing on the conserved N-terminal acetyltransferases (NATs) family, we revealed diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We demonstrated that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harbouring hotspots exhibited high levels of thermo-lability, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complex assembly. We next demonstrated that this assembly disruption led to subunits aggregation, analysed by advanced imaging approaches. Accordingly, conservation analysis revealed that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include more complexes, we find similar trends, allowing us to propose a predictive model.
To address aim (iii), we developed a single-molecule ribosome profiling approach, termed RiboFriend. We relied on our experience in combining biochemistry and sequencing to develop a nanopore based approach, to capture the interplay of factors guiding co-translational folding, in vivo. We have generated two chimeric constructs, fusing each co-translationally acting factor with a different RNA modifying enzyme. Direct RNA sequencing using NanoPore revealed that over 700 RNA transcripts exhibit significant mRNA modifications.
The ERC grant has enabled us several significant breakthroughs in this time period.
In aim (i) we have identified novel ribosome-interacting proteins, significantly enriched in our pull-down of ribosomes synthesizing misfolding-prone subunits. We have detected dozens of proteins belonging to the ubiquitin-proteasome pathway. Furthermore, we have discovered several putative E3 and molecular chaperones, which to date have not been characterized. Many of them share high sequence conservation to mammalian. These results demonstrate for the first time that inhibition of co-translational assembly pathways lead to induction of dedicated co-translational quality control pathways.
In aim (ii) We performed selective ribosome profiling analysis with extensive molecular dynamics simulations. We identified critical amino acids as well as structural elements critical for complex formation of the NATs family (N-Acetyl-Transferases). These features were also found to stabilize and facilitate the folding of the nascent chain, as it emerges from the ribosome. The simulations-based predictions were then tested and validated in vivo by various approaches, capturing the translating ribosomes in the cells, and the impact of disrupting co-translational assembly pathways. We extended our exploration to include several other complexes. Combining modelling with molecular dynamics simulation, we derived interface energy profiles. Our findings demonstrated the predictive power of our combined approaches to identify the critical residues for co-translational interactions. This is a paradigm shift, that exposure of these anchors at the ribosome exit tunnel can direct the entire polypeptide folding and assembly into functional complex. Conservation analysis revealed disease-related mutations may function as such anchors, suggesting therapeutic targets.
In aim (iii) we developed RiboFriend, a single molecule approach for in vivo analysis of co-translational factors interplay. We revealed the interplay of two canonical ribosome-associated chaperones on a single transcript. Furthermore, the results already demonstrate the power of the newly developed RiboFriend tool to resolve long standing questions in the rapidly evolving field of protein synthesis.
Taken together, the results suggest the ERC grant output can revolutionize our understanding of the interplay between co-translational protein folding and misfolding. The emphasis on early events, occurring during protein synthesis, can pave the way for development of preventive strategies, providing novel therapeutic targets for disease conditions characterized by protein misfolding, such as Parkinson's and Alzheimer's.
In aim (i) we have identified novel ribosome-interacting proteins, significantly enriched in our pull-down of ribosomes synthesizing misfolding-prone subunits. We have detected dozens of proteins belonging to the ubiquitin-proteasome pathway. Furthermore, we have discovered several putative E3 and molecular chaperones, which to date have not been characterized. Many of them share high sequence conservation to mammalian. These results demonstrate for the first time that inhibition of co-translational assembly pathways lead to induction of dedicated co-translational quality control pathways.
In aim (ii) We performed selective ribosome profiling analysis with extensive molecular dynamics simulations. We identified critical amino acids as well as structural elements critical for complex formation of the NATs family (N-Acetyl-Transferases). These features were also found to stabilize and facilitate the folding of the nascent chain, as it emerges from the ribosome. The simulations-based predictions were then tested and validated in vivo by various approaches, capturing the translating ribosomes in the cells, and the impact of disrupting co-translational assembly pathways. We extended our exploration to include several other complexes. Combining modelling with molecular dynamics simulation, we derived interface energy profiles. Our findings demonstrated the predictive power of our combined approaches to identify the critical residues for co-translational interactions. This is a paradigm shift, that exposure of these anchors at the ribosome exit tunnel can direct the entire polypeptide folding and assembly into functional complex. Conservation analysis revealed disease-related mutations may function as such anchors, suggesting therapeutic targets.
In aim (iii) we developed RiboFriend, a single molecule approach for in vivo analysis of co-translational factors interplay. We revealed the interplay of two canonical ribosome-associated chaperones on a single transcript. Furthermore, the results already demonstrate the power of the newly developed RiboFriend tool to resolve long standing questions in the rapidly evolving field of protein synthesis.
Taken together, the results suggest the ERC grant output can revolutionize our understanding of the interplay between co-translational protein folding and misfolding. The emphasis on early events, occurring during protein synthesis, can pave the way for development of preventive strategies, providing novel therapeutic targets for disease conditions characterized by protein misfolding, such as Parkinson's and Alzheimer's.