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Regulated Proteolysis: The Secrets of Lon: Cellular Roles and Stress-dependent Regulation of the Lon Protease
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-4620-2009
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteolysis is crucial in cells to maintain a functional proteome. It is required for removal of damaged and unfolded proteins during protein quality control, and serves as a mechanism to regulate protein levels through regulated proteolysis. The latter targets specific proteins under certain conditions to rapidly adjust the levels of these proteins. Thereby, activity of the targeted proteins is reduced or even eliminated. Many cellular processes like cell cycle progression, differentiation and stress response/adaptation depend on proteolytic removal of regulatory proteins via proteolysis. One protease with crucial roles in both protein quality control and regulated proteolysis is called Lon and is found in many species across all domains of life.

Extensive research has revealed many aspects of proteolysis by Lon and identified several Lon substrates. The dimorphic bacterium Caulobacter crescentus is a model organisms to study proteolysis and Lon as the two cell types, a flagellated swarmer cell and a stalked sessile cell, and the transition between them heavily depends on proteolysis. However, certain details, like recognition of substrates and its regulation are largely unknown. Here I focus on regulated proteolysis by Lon in C. crescentus, specifically on novel substrates, their recognition, and the regulation of Lon.

Study I: Using quantitative proteomics of wildtype and mutant C. crescentus strains we identified potential substrates of Lon. Out of these, we focused on the stalk biogenesis regulator StaR and the flagellar hook length determination factor FliK. Both proteins are developmental regulators, whose protein levels oscillate during the cell cycle. Our experiments showed that turnover by Lon is required to maintain these oscillations and disruption thereof results in deregulation of the stalk and the flagellum.

Study II: We used proteolytically inactive Lon to co-purify interactors of Lon and identify them by mass spectrometry. Thereby, we found an uncharacterized heat shock protein that regulates the activity of Lon, and due to our findings named it LarA (Lon activity regulator A). We showed that LarA interacts with Lon at an allosteric site and modulates the activity of Lon via its C-terminal amino acids. In most cases LarA exhibits a stimulating effect on the degradation of the substrates, indicating that LarA regulates substrate specificity and guarantees efficient degradation of the affected substrates. The same residues involved in the modulating interaction also serve as a degron for degradation of LarA by Lon to shut off LarA-mediated modulation if not needed anymore.

Study III: SigT is a driver of gene expression of the general stress response in C. crescentus. It was detected in both previous studies, which indicates that it is degraded by Lon. We showed that it is a substrate of Lon in vitro. Based on steady-state levels of SigT during and after sucrose-induced stress, we could show that Lon-mediated degradation is important during the recovery. In addition, LarA-mediated regulation of Lon stimulates turnover of SigT, indicating that SigT levels are fine-tuned by LarA under certain conditions.

In summary, we identified novel regulatory roles of Lon on differentiation and stress response in C. crescentus and discovered LarA as a novel modulator of Lon activity. The results of these studies once more emphasize the importance of Lon as a regulator of various cellular processes. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2023. , p. 63
Keywords [en]
Lon protease, regulated proteolysis, allosteric regulation, Caulobacter crescentus, stress responses
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-214634ISBN: 978-91-8014-192-5 (print)ISBN: 978-91-8014-193-2 (electronic)OAI: oai:DiVA.org:su-214634DiVA, id: diva2:1735060
Public defence
2023-03-23, P216, NPQ-huset, Svante Arrhenius Väg 20 A, Stockholm, 13:30 (English)
Opponent
Supervisors
Available from: 2023-02-28 Created: 2023-02-07 Last updated: 2023-02-28Bibliographically approved
List of papers
1. The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle
Open this publication in new window or tab >>The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle
2021 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e73875Article in journal (Refereed) Published
Abstract [en]

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Delta lon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Keywords
Lon protease, proteolysis, cell differentiation, flagella, FliK, bacterial cell cycle, Other
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-198759 (URN)10.7554/eLife.73875 (DOI)000711524700001 ()34693909 (PubMedID)
Available from: 2021-11-17 Created: 2021-11-17 Last updated: 2023-02-07Bibliographically approved
2. The heat shock protein LarA activates the Lon protease at the onset of proteotoxic stress
Open this publication in new window or tab >>The heat shock protein LarA activates the Lon protease at the onset of proteotoxic stress
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that LarA is regulated by Lon itself, which is critical to prevent toxic overactivation of Lon. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand at the onset of protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.

Keywords
proteolysis, protein quality control, allosteric regulation, bacteria, heat shock response, AAA+ protease
National Category
Biological Sciences
Research subject
Molecular Bioscience; Molecular Biology; Microbiology
Identifiers
urn:nbn:se:su:diva-214632 (URN)10.1101/2022.10.10.511565 (DOI)
Available from: 2023-02-07 Created: 2023-02-07 Last updated: 2023-02-07
3. Regulation of the ECF sigma factor SigT by Lon-mediated proteolysis
Open this publication in new window or tab >>Regulation of the ECF sigma factor SigT by Lon-mediated proteolysis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Lon protease is widely conserved in both prokaryotic and eukaryotic species in which it fulfils important regulatory functions. Nevertheless, the number of identified Lon substrates is limited in most organisms and the precise role of Lon in regulating these proteins poorly understood. Previous quantitative proteomics data classified the general stress response sigma factor SigT as a promising putative Lon substrate in the dimorphic bacterium Caulobacter crescentus. Here, we confirm that SigT abundance is directly regulated by Lon. We show that downregulation of SigT levels during recovery from sucrose-induced osmotic stress is delayed in the absence of Lon, while its upregulation at the onset of stress functions normally. Furthermore, the presence of the Lon regulator LarA enhances Lon-mediated degradation of SigT in vitro and reduces SigT levels in vivo indicating a role of LarA in regulating Lon-mediated degradation of SigT. Together, our results emphasize the importance of Lon during the recovery phase following stress exposure by adjusting the concentration of the general stress response regulator SigT.

Keywords
Lon protease, stress response, ECF sigma factor, proteolysis
National Category
Biological Sciences
Research subject
Molecular Biology; Molecular Bioscience; Molecular Biology
Identifiers
urn:nbn:se:su:diva-214633 (URN)
Available from: 2023-02-07 Created: 2023-02-07 Last updated: 2023-02-07

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