| 1. |
- Giovannucci, Tatiana A., et al.
(författare)
-
Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy
- 2022
-
Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 18:7, s. 1486-1502
-
Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.
|
|
| 2. |
- Leckey, Claire A, et al.
(författare)
-
CSF neurofilament light chain profiling and quantitation in neurological diseases.
- 2024
-
Ingår i: Brain communications. - 2632-1297. ; 6:3
-
Tidskriftsartikel (refereegranskat)abstract
- Neurofilament light chain is an established marker of neuroaxonal injury that is elevated in CSF and blood across various neurological diseases. It is increasingly used in clinical practice to aid diagnosis and monitor progression and as an outcome measure to assess safety and efficacy of disease-modifying therapies across the clinical translational neuroscience field. Quantitative methods for neurofilament light chain in human biofluids have relied on immunoassays, which have limited capacity to describe the structure of the protein in CSF and how this might vary in different neurodegenerative diseases. In this study, we characterized and quantified neurofilament light chain species in CSF across neurodegenerative and neuroinflammatory diseases and healthy controls using targeted mass spectrometry. We show that the quantitative immunoprecipitation-tandem mass spectrometry method developed in this study strongly correlates to single-molecule array measurements in CSF across the broad spectrum of neurodegenerative diseases and was replicable across mass spectrometry methods and centres. In summary, we have created an accurate and cost-effective assay for measuring a key biomarker in translational neuroscience research and clinical practice, which can be easily multiplexed and translated into clinical laboratories for the screening and monitoring of neurodegenerative disease or acute brain injury.
|
|
| 3. |
- Giovannucci, Tatiana A
(författare)
-
Tipping the scales in cancer : novel mechanisms of inhibiting protein degradation
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein homeostasis (in short, ‘proteostasis’) requires the timely degradation of proteins to retain control on protein quality, amount and function. Two main proteolytic systems, the ubiquitin-proteasome system (UPS) and autophagy, complement each other to fulfill this regulatory role. Defective proteostasis is linked prominently to age-related disease, including neurodegenerative disorders and cancer. Components of the UPS and autophagy are often mutated or dysregulated during cancer progression, a phenomenon linked to sustained cell proliferation, tumor growth and resistance to therapy. Hence, cancer cells display an increased sensitivity towards drugs that reduce the function of these proteolytic systems, as illustrated by the clinical success of inhibitors of the proteasome, and the clinical trials of lysosome neutralizers to inhibit autophagy in cancer. However, severe side effects, therapy-induced resistance and a lack of efficacy hamper their use, underscoring the need for more effective and tumor-selective compounds blocking the UPS and autophagy. The work presented in this thesis set out to identify novel inhibitors of the UPS. Despite being rich in potentially druggable proteins, it is challenging to identify, a priori, a suitable target for drug development due to extensive functional redundancy across the pathway. Thus, we took two different approaches to find novel mechanisms for inhibition of protein degradation. In papers I and II, we used a cancer cell line stably expressing a fluorescent UPS reporter and employed a forward chemical genetic screening approach to interrogate the pathway in an unbiased manner, seeking new targets and/or new therapeutics to inhibit proteolysis in cancer. In paper III, we explored whether inhibiting the turnover of specific UPS reporters can be achieved by modulating the delivery of proteins to the proteasome. In paper I, we characterized CBK77, a first-in-class UPS inhibitor that requires the enzymatic activity of the oxidoreductase NQO1 to be activated in cells. CBK77 impairs the degradation of ubiquitin-dependent substrates, leading to the accumulation of ubiquitylated proteins followed by caspase-mediated cell death. We found that activated CBK77 binds to ubiquitin and hinders deubiquitylating activity in vitro, providing a plausible mechanism for CBK77-induced UPS impairment. We propose that bioactivation can be exploited as a new means to increase cancer selectivity of UPS inhibitors. In paper II, we describe CBK79, a promising novel small molecule inhibitor of proteostasis that simultaneously impairs both the UPS and autophagy and induces profound proteotoxic stress in cancer cells. Whilst this leads to the activation of several stress responses to counteract the disruptive effect of CBK79 on proteolysis, these are not successful in restoring homeostasis or preventing cell death. This work shows the potential of dual targeting of the UPS and autophagy for the development of inhibitors that can overcome cellular compensatory mechanisms and could therefore result in more efficient targeting of cancer cells. In paper III, we studied the mechanisms controlling the delivery of proteins to the proteasome through the ubiquitin-dependent Cdc48/VCP/p97 pathway. We used the turnover of Cdc48-dependent fluorescent reporters in yeast to study these processes and found that the ubiquitin shuttle protein Rad23 is itself ubiquitylated prior to substrate delivery. Modulating this step affected the degradation of Cdc48 model substrates. Overall, our findings reveal an additional layer of regulation in the UPS that could be explored for pharmacological intervention. In conclusion, by employing reporter substrates of the UPS, we have uncovered new compounds and highlighted novel regulatory processes amenable to targeting with the ultimate goal of expanding the army of proteolysis inhibitors with anti-cancer properties.
|
|
