The consecutive actions of the ubiquitin-selective segregase Cdc48 and the ubiquitin shuttle factor Rad23 result in the delivery of ubiquitylated proteins at the proteasome. Here, we show that the deubiquitylating enzyme Ubp12 interacts with Cdc48 and regulates proteasomal degradation of Rad23-dependent substrates in Overexpression of Ubp12 results in stabilization of Rad23-dependent substrates. We show that Ubp12 removes short ubiquitin chains from the N-terminal ubiquitin-like domain (UbL) of Rad23. Preventing ubiquitylation of Rad23 by mutation of lysine residues within the UbL domain, Rad23, does not affect the non-proteolytic role of Rad23 in DNA repair but causes an increase in ubiquitylated cargo bound to the UBA2 domain of Rad23, recapitulating the stabilization of Rad23-dependent substrates observed upon overexpression of Ubp12. Expression of Rad23 or overexpression of Ubp12 impairs the ability of yeast to cope with proteotoxic stress, consistent with inefficient clearance of misfolded proteins by the ubiquitin-proteasome system. Our data suggest that ubiquitylation of Rad23 plays a stimulatory role in the degradation of ubiquitylated substrates by the proteasome.
Instant and adequate handling of misfolded or otherwise aberrant proteins is of paramount importance for maintaining protein homeostasis in cells. The ubiquitin/proteasome system (UPS) is a central player in protein quality control as it operates in a seek-and-destroy mode, thereby facilitating elimination of faulty proteins. While proteasome inhibition is in clinical use for the treatment of hematopoietic malignancies, stimulation of the UPS has been proposed as a potential therapeutic strategy for various neurodegenerative disorders. High-throughput screens using genetic approaches or compound libraries are powerful tools to identify therapeutic intervention points and novel drugs. Unlike assays that measure specific activities of components of the UPS, reporter substrates provide us with a more holistic view of the general functional status of the UPS in cells. As such, reporter substrates can reveal new ways to obstruct or stimulate this critical proteolytic pathway. Here, we discuss various reporter substrates for the UPS and their application in the identification of key players and the pursuit for novel therapeutics.
Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.
BackgroundNeurofilament light polypeptide (NfL) is a basic component of intermediate filaments in neurons that is released from damaged and/or degenerated neuronal axons. As such, NfL levels in CSF and serum are elevated in several neurodegenerative diseases, and have been proposed as a promising biomarker of axonal damage. However, whether NfL’s extracellular release results from passive events or active mechanisms remains largely unknown. Kinetic labeling at the step of protein translation can inform about active translation vs. passive release processes and provide protein metabolism half‐lives and rates of production and clearance.MethodOur aim was to develop a method to study NfL turnover rates using stable isotope labeling kinetics (SILK) in human induced pluripotent stem cell (iPSC)‐derived neurons. We added 13C6‐leucine, the tracer, to the neuron cultures (“pulse”) for four weeks, followed by medium exchange with 12C6‐leucine, the tracee, for an additional four weeks (“chase”). Samples were collected during both the pulse and chase phases of the experiment. 13C6‐leucine incorporation and the levels of labeled NfL in cell lysates and conditioned media from two independent neuronal inductions were analyzed by ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) and the tracer‐to‐tracee ratio (TTR) and mole percent excess (MPE) calculated.ResultWe detected ten leucine‐containing peptides unique to NfL in neuronal cell lysates, which are present within the rod‐domain of the protein. These peptides showed different rates of 13C6‐leucine incorporation (reaching up to 69% MPE).ConclusionPreliminary data on the monitoring of these peptides suggest that NfL is turned over at a slow rate in iPSC‐. Tracking of both intracellular and extracellular NfL in neurons derived from healthy control individuals, as well as cells with mutations implicated in neurodegenerative diseases, are expected to shed light into the dynamics of NfL turnover in both physiological and pathological conditions. This will be critical for interpreting NfL in response to disease‐modifying therapies.
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.
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