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Unfoldomics of the cellular stress response: How do intrinsically disordered chaperones work?

Final Report Summary - IDCHAPERONES (Unfoldomics of the cellular stress response: How do intrinsically disordered chaperones work?)

Proteins are the most diverse and structurally complex macromolecules in the cell. Considered Nature's workhorses, they are involved in almost every aspect of biological function known. The function of proteins is defined by their specific three-dimensional structure, making it a top priority for every cell and organism to ensure that nascent polypeptide chains adopt and native proteins preserve their properly folded conformation. Control over a healthy proteome (i.e. proteostasis) begins with the birth of the polypeptide chain on the ribosome and ends with the coordinated death of the mature protein by degradation. Each step in between is carefully orchestrated and involves a complex and highly dynamic network of proteostasis factors. Failure of this network may lead to the toxic accumulation of misfolded proteins and pathologies.
One of the major players of the proteostatic network are molecular chaperones. Molecular chaperones prevent unfavorable interactions between protein folding intermediates, which would otherwise lead to irreversible and cytotoxic aggregate formation. Under stress conditions such as heat, oxidative and acidic stresses, many proteins unfold, potentially leading to aggregation and subsequently to cell death. It is therefore not surprising that many molecular chaperones are constitutively expressed during non-stress conditions and are massively overexpressed under stress conditions that lead to protein unfolding.
Molecular chaperones are divided into two main mechanistic classes: ATP-dependent foldases and ATP-independent holdases (or holding chaperones). Foldases (e.g. the prokaryotic DnaK-system, eukaryotic Hsp90, Hsp70) actively participate in protein folding processes during non-stress and stress conditions. However, ATP-independent molecular chaperones, holdases, usually are stress-dependent and serve as the "first responders" of the protein homeostasis system during specific stress condition. Holdases are equipped with highly specific stress sensing mechanisms, which enables them to rapidly respond to distinct stress conditions (e.g. Hsp33 upon exposure to oxidative unfolding. sHsp during heat conditions).
Working cycle of many of the ATP-independent chaperones relays on structural plasticity of the chaperone, which is usually controlled by post-translational events and is stress-specific (e.g oxidation of redox-sensitive cysteines in oxidation stress specific chaperone, Hsp33, protonation of specific residues in acidic stress specific chaperone, HdeA). This structural plasticity is encoded in intrinsically disordered regions of the chaperones.
In this study we aim to answer the following questions: What are the sequence features that define ATP-independent chaperones which allows their activation and function? Are these sequences different from other unfolded or intrinsically disordered proteins, with no chaperone activity? Can we predict novel intrinsically disordered chaperones? What methodologies can be used to map conformational changes of intrinsically disordered chaperones during their working cycle and understand their role in chaperone activity.
To answer on these questions, we utilized interdisciplinary techniques and combined computational biology with proteins design and chaperone biochemistry. To understand role and sequence features of intrinsically disordered regions in chaperone function, we used redox-regulated chaperone, Hsp33, as a model protein. Moreover, to extend knowledge about identity, function and role of intrinsically disordered regions in chaperone function we established cutting-age technologies based on mass spectrometry to identify in-vivo interactome of thermobile protein, to map interaction sites and examine conformational changes along working cycle of chaperones.
Our achievements are:
1. Characterization of role and sequence properties of thermobile regions of Hsp33. We found that the intrinsically disordered region of Hsp33 serves as an anchor for the reduced, inactive state of Hsp33, and it dramatically affects the crosstalk with the synergetic chaperone system, DnaK/J. Moreover, we found that Hsp33 interacts with non-stress members of the proteostatic bacterial network, and that this interaction, probably, does not involve thermobile regions of Hsp33.
2. Identification and characterization of Hsp33 homologue in eukaryotic Trypanosoma brucei pathogen, that causes 'sleeping sickness' in humans, which is responsible for thousands of deaths every year.
3. Establishment a toolbox of cutting-edge mass spectrometry technology to characterize interactome in-vivo, and to map conformational changes in proteins upon interactions and/or post-translational modifications, specifically focusing on molecular chaperones.
This project paved new avenues in the research of ATP-independent chaperones and stress response as well as promoted development of cutting age technologies. On the personal note, this project promoted my professional carrier and enable establishing new collaborations, publish and present this work in international meetings.