The ubiquitin–proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.
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Bacterial degrons in synthetic circuits
Bacterial proteases are a promising post-translational regulation strategy in synthetic circuits because they recognize specific amino acid degradation tags (degrons) that can be fine-tuned to modulate the degradation levels of tagged proteins. For this reason, recent efforts have been made in the search for new degrons. Here we review the up-to-date applications of degradation tags for circuit engineering in bacteria. In particular, we pay special attention to the effects of degradation bottlenecks in synthetic oscillators and introduce mathematical approaches to study queueing that enable the quantitative modelling of proteolytic queues.
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- PAR ID:
- 10426750
- Date Published:
- Journal Name:
- Open Biology
- Volume:
- 12
- Issue:
- 8
- ISSN:
- 2046-2441
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.
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The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome’s ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub 4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub 4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates while the Ub 4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.more » « less
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Abstract There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplify reporter signal and permanently mark all descendants. In addition, we present a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improve the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. Although each promoter requires tuning for optimal performance, this integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.
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Abstract Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the Homology Region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the Leucine-Rich Repeat (LRR) and novel E3 ligase (NEL) domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high temporal resolution and may become an important tool in plant cell biology.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1098/rsob.220180
