Diverging co-translational protein complex assembly pathways are governed by interface energy distribution

Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find 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 harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes' complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.This work was funded by the European Research Council Starting Grant 2031817 (A.S.) and the Israeli Science Foundation grants 2106/20 (A.S.) and 1515/23 (O.K.). This work was also partially supported by grant number PID2022-143215OB-I00, funded by MCIN/AEI/10.13039/501100011033/ FEDER, UE (J.F.R).Peer reviewe

A European network to fine-tune the proteome

Publisher Copyright: © 2024 The Author(s)Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).publishersversioninpres

Chaperoning Proteins for Destruction: Diverse Roles of Hsp70 Chaperones and their Co-Chaperones in Targeting Misfolded Proteins to the Proteasome

Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases

Yeast: Translation Regulation and Localized Translation

Translation regulation and localized translation are essential for protein synthesis, controlling all aspects of cellular function in health and disease [...

Flow cytometric quantification and characterization of intracellular protein aggregates in yeast

<div><p>The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.</p></div

Diverging co-translational protein complex assembly pathways are governed by interface energy distribution

Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find 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 harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes' complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.This work was funded by the European Research Council Starting Grant 2031817 (A.S.) and the Israeli Science Foundation grants 2106/20 (A.S.) and 1515/23 (O.K.). This work was also partially supported by grant number PID2022-143215OB-I00, funded by MCIN/AEI/10.13039/501100011033/ FEDER, UE (J.F.R).Peer reviewe

30 years of ZMBH

Mit einem Festakt und einem Alumni-Forum begeht das ZMBH am 17. und 18. September 2015 sein 30-jähriges Bestehen. An dieser zentralen Forschungseinrichtung der Ruperto Carola widmen sich Wissenschaftler verschiedener Disziplinen der Erforschung grundlegender molekular- und zellbiologischer Prozesse, die vielfach auch medizinische Aspekte berühren

ProteoCure : A European network to fine-tune the proteome

International audienceProteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion.Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential.The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s)