| 1. |
- Heessen, Stijn
(författare)
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Regulation of the ubiquitin-proteasome system : characterization of viral and cellular stabilization signals
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ubiquitin-proteasome system plays a fundamental role in virtually every cellular process. Degradation of endogenous proteins by this system is the major source for antigenic peptides that are presented to MHC class I-restricted cytotoxic T cells. The, Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) contains a long glycine-alanine (GA) repeat that inhibits proteasomal processing, resulting in a blockage of antigen presentation. The GA repeat acts in cis and can be functionally transferred to other proteins. The aim of the work described in this thesis was to characterize the protective effect of this viral stabilization signal and to identify similar cellular stabilization signals. To evaluate the inhibitory activity of the GA repeat, we used green fluorescent protein (GFP)-based reporters that were targeted for ubiquitin/proteasome-dependent proteolysis by various degrons. Introducing GA repeats of increasing length resulted in enhanced protection of the fluorescent reporter from proteolysis. When provided with a strong degradation signal even EBNA1 could be efficiently degraded. This study showed that a balance between the strength of the degradation signal and the length of the repeat determines the GA repeat-dependent stabilization effect. Next, we tested the ability of the GA repeat to prevent degradation of the tumor suppressor p53 because inactivation of p53 by accelerated degradation is a common event in tumor development. P53-GA repeat chimeras were protected from degradation and showed improved growth inhibitory activity in tumor cells with impaired endogenous p53 activity, suggesting that insertion of the GA repeat could provide a convenient strategy for the stabilization of potential therapeutic proteins. We used the aforementioned GFP reporters to test the protective effect of the GA repeat in the yeast Saccharomyces cerevisiae. Expression of proteins carrying GA repeats required the generation of yeast codon-optimized recombinant GA (rGA) repeats. We found that introduction of rGA repeats in the GFP substrates resulted in stabilization of the proteins in mammalian and yeast cells, indicating that the protective signal targets a conserved mechanism in the ubiquitin-proteasome system. The yeast DNA-repair protein Rad23 is long-lived despite the fact that it is ubiquitinated and interacts with the proteasome. We investigated whether Rad23 contains domains that can protect it from proteasomal degradation. Disruption of the UBA2 domain converted Rad23 into a short-lived protein that is targeted for proteasomal degradation through its ubiquitin-like domain. Insertion of the UBA2 domain from Rad23 or its human homologue HHR23A prevented the degradation of destabilized GFP reporters without causing a general inhibition of proteolysis. We suggest that the Rad23 UBA2 domain functions as a novel cis-acting stabilization signal that confers protection against proteasomal degradation.
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| 2. |
- Martins, António, 1976-, et al.
(författare)
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Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling protease
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5)-sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy-terminal of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Δtether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non- and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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| 3. |
- Martins, António, et al.
(författare)
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Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor-controlled Ssy5 signaling protease
- 2019
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 30:21, s. 2709-2720
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5) sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy terminus of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Delta tether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution, and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non-and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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| 4. |
- Martins, António, et al.
(författare)
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Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity
- 2018
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 293:22, s. 8362-8378
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Tidskriftsartikel (refereegranskat)abstract
- Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.
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| 5. |
- Pfirrmann, Thorsten, et al.
(författare)
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The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp1
- 2010
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 30:13, s. 3299-309
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells
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| 6. |
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