Nt-acetylation independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases
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PROVIDER: PRJNA989617 | ENA |
REPOSITORIES: ENA
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Project description:The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3-ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharmoyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localised squalene epoxidase 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.
2023-11-13 | GSE236282 | GEO
Project description:The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by specific E3-ligases (Ac/N-recognins). To date, no Ac/N-recognins have been defined in plants. Here we use molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis DOA10-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no significant differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we show that turnover of ER-localised squalene epoxidase 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta does not depend on NTA, but Nt-acetyltransferases indirectly impact its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis, and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.
2024-01-26 | PXD043217 | Pride
Project description:The Hace1 E3 ligase is a tumor suppressor in stressed cells. Through unknown mechanisms, Hace1 indirectly targets the cyclin D1 proto-oncogene for proteasomal degradation during nutrient depletion. We now show that Hace1 targets HIF1alpha for VHL-dependent degradation during hypoxia. To better understand these diverse actions we performed mass spectrometry to identify Hace1-interacting proteins. We show that Hace1 interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1) under nutrient depletion and hypoxia. CAND1 binds cullins and prevents their entry into cullin ring E3 ligase (CRL) complexes, thus blocking CRL activity. Hace1 binding releases CUL1/2 from CAND1, facilitating assembly of CRL complexes to degrade cyclin D1 and HIF1alpha, respectively. These findings suggest a broad role for Hace1 in regulating tumor suppressive CRL E3 ligases. In this study, we used gene expression profiling to characterize how Hace1 overexpression affect the transcriptional response to hypoxic stress Using Affymetrix exon-level microarrays, we compared the expression profile of HEK293 cells overexpressing either Hace1 or MSCV vector alone, under hypoxia or normoxia
2011-07-01 | E-GEOD-25452 | biostudies-arrayexpress
Project description:SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression
| PRJNA742677 | ENA
Project description:The majority of current therapeutics targeting plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. Typical mammalian proteins, however, consist of multiple domains executing discrete but coordinated activities, and saturating inhibition of one functional domain often incompletely suppresses the totality of the protein’s function. Recent work on targeted protein degradation technologies including Proteolysis Targeting Chimeras (PROTACs) has highlighted clinically important distinctions between target inhibition and target degradation. However, the generation of heterobifunctional compounds requiring linkage of two small molecules, each with high affinity for their targets, is highly complex, particularly with respect to achieving oral bioavailability. Here we describe the development of Proteolysis Targeting Antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target ubiquitination and subsequent degradation. PROTAB-mediated degradation drives deeper pathway inhibition than inhibitory antibodies and is functional in vivo. The scope of this technology is also demonstrated through the identification of additional cell surface E3 ubiquitin ligases that can function as “on demand” degraders of various cell surface proteins. The generality of this approach enables tissue-selective degradation, as suggested by the Wnt-responsive ligases RNF43 and ZNRF3. Furthermore, through engineering of various optimized antibody formats, we offer insights on the ground rules governing optimal target degradation. Taken together, this work describes a strategy for the rapid development of potent, bioavailable and tissue selective degradation of cell surface proteins.
2022-07-21 | GSE208372 | GEO
Project description:The Hace1 E3 ligase is a tumor suppressor in stressed cells. Through unknown mechanisms, Hace1 indirectly targets the cyclin D1 proto-oncogene for proteasomal degradation during nutrient depletion. We now show that Hace1 targets HIF1alpha for VHL-dependent degradation during hypoxia. To better understand these diverse actions we performed mass spectrometry to identify Hace1-interacting proteins. We show that Hace1 interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1) under nutrient depletion and hypoxia. CAND1 binds cullins and prevents their entry into cullin ring E3 ligase (CRL) complexes, thus blocking CRL activity. Hace1 binding releases CUL1/2 from CAND1, facilitating assembly of CRL complexes to degrade cyclin D1 and HIF1alpha, respectively. These findings suggest a broad role for Hace1 in regulating tumor suppressive CRL E3 ligases. In this study, we used gene expression profiling to characterize how Hace1 overexpression affect the transcriptional response to hypoxic stress
2011-07-01 | GSE25452 | GEO
Project description:The functionally redundant ubiquitin E3 ligases SIAH1 and SIAH2 have been implicated in the regulation of several processes, but their role in inflammatory signaling and gene expression remains unclear. Here we have downregulated the expression of both SIAH proteins with specific siRNAs and investigated the functional consequences for IL-1α-induced gene expression and the signaling pathways activating NF-κB and AP1. The knockdown of SIAH1/2 had no significant impact on IL-1α-induced activation of NF-κB and MAPK signaling pathways, but modulated the expression of approximately one third of IL-1α-regulated genes. Most of the proteins encoded by SIAH1/2-regulated genes form a regulatory network of proinflammatory factors. Thus SIAH1/2 proteins function as variable rheostats that control the amplitude of inflammatory gene expression.
2022-02-01 | GSE179190 | GEO
Project description:Paeonia lactiflora restored skeletal muscle function and mass in cancer-bearing mice, through the downregulations of muscular NF-kB signalling and muscle-specific E3 ubiquitin ligases. This study provides experimental evidence that Paeonia lactiflora can be a plausible candidate for a therapeutic agent for cancer cachexia.
2019-06-04 | GSE132120 | GEO
Project description:Repurposing E3 ubiquitin ligases as cell surface protein degraders using Proteolysis Targeting Antibodies
| PRJNA859706 | ENA
Project description:The post-translational modification of proteins by ubiquitination is a highly regulated process that involves a dynamic, three-step enzymatic cascade, where more than 600 E3 ligases play a critical role in recognizing specific substrates for modification. Separating bona fide targets of E3s from E3-interacting proteins remains a major challenge in the field. In this study, we present BioE3, a novel approach for identifying substrates of ubiquitin-like (UbL) E3 ligases of interest. Using BirA-E3 ligase fusion proteins and bioUbLs, the method facilitates site-specific biotinylation of UbL-modified substrates for proteomic identification. We demonstrate that the BioE3 system can identify both known and novel targets of two RING-type ubiquitin E3 ligases: RNF4, known to be involved in DNA damage response and the regulation of PML nuclear bodies, and MIB1, implicated in endocytosis, autophagy, and centrosomal protein homeostasis. We further show the versatility of BioE3 by identifying targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, we show that BioE3 works with HECT-type E3 ligases and identify novel targets of NEDD4 involved in vesicular trafficking. BioE3 is a powerful tool that enables identification of bona fide substrates of UbL E3 ligases and how they change with chemical perturbations, which may be useful for the emerging applications in targeted protein degradation (TPD). BioE3 may also be applicable for UbLs beyond Ub and SUMO, as well as other E3 ligase classes. The resulting knowledge can shed light on the regulation of cellular processes by the complex UbL network, advancing our understanding of fundamental biological mechanisms.
2024-01-26 | PXD041685 | Pride