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The Plant Cell Sep 2023Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48...
Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.
Topics: Arabidopsis; Arabidopsis Proteins; Cold-Shock Response; Polyubiquitin; Transcription Factors; Ubiquitination; Ubiquitin
PubMed: 37279565
DOI: 10.1093/plcell/koad159 -
Nature Mar 2024Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target...
Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
Topics: Bromodomain Containing Proteins; Cell Cycle Proteins; Drug Design; Proteasome Endopeptidase Complex; Proteolysis; Proteolysis Targeting Chimera; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitination; Protein Binding; Substrate Specificity; Protein Domains
PubMed: 38383787
DOI: 10.1038/s41586-024-07089-6 -
Journal of Cachexia, Sarcopenia and... Oct 2023Corylifol A (CYA) is one of the main active components of Psoralea corylifolia L. CYA had been reported to have ameliorating effects on dexamethasone-induced atrophy of...
BACKGROUND
Corylifol A (CYA) is one of the main active components of Psoralea corylifolia L. CYA had been reported to have ameliorating effects on dexamethasone-induced atrophy of C2C12 mouse skeletal myotubes, but its effects on cancer cachexia were unclear. Here, we checked the influence of CYA on muscle atrophy in cancer cachexia mice and tried to clarify its mechanisms.
METHODS
C26 tumour-bearing mice were applied as the animal model to examine the effects of CYA in attenuating cachexia symptoms. The in vitro cell models of TNF-α-induced C2C12 myotubes or ad-mRFP-GFP-LC3B-transfected C2C12 myotubes were used to check the influence of CYA on myotube atrophy based on both ubiquitin proteasome system (UPS) and autophagy-lysosome system. The possible direct targets of CYA were searched using the biotin-streptavidin pull-down assay and then confirmed using the Microscale thermophoresis binding assay. The levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting and immunocytochemical assay.
RESULTS
The administration of CYA prevented body weight loss and muscle wasting in C26 tumour-bearing mice without affecting tumour growth. At the end of the experiment, the body weight of mice treated with 30 mg/kg of CYA (23.59 ± 0.94 g) was significantly higher than that of the C26 model group (21.66 ± 0.56 g) with P < 0.05. The values of gastrocnemius muscle weight/body weight of mice treated with 15 or 30 mg/kg CYA (0.53 ± 0.02% and 0.54 ± 0.01%, respectively) were both significantly higher than that of the C26 model group (0.45 ± 0.01%) with P < 0.01. CYA decreased both UPS-mediated protein degradation and autophagy in muscle tissues of C26 tumour-bearing mice as well as in C2C12 myotubes treated with TNF-α. The thousand-and-one amino acid kinase 1 (TAOK1) was found to be the direct binding target of CYA. CYA inhibited the activation of TAOK1 and its downstream p38-MAPK pathway thus decreased the level and nuclear location of FoxO3. siRNA knockdown of TAOK1 or regulation of the p38-MAPK pathway using activator or inhibitor could affect the ameliorating effects of CYA on myotube atrophy.
CONCLUSIONS
CYA ameliorates cancer cachexia muscle atrophy by decreasing both UPS degradation and autophagy. The ameliorating effects of CYA on muscle atrophy might be based on its binding with TAOK1 and inhibiting the TAOK1/p38-MAPK/FoxO3 pathway.
PubMed: 37439183
DOI: 10.1002/jcsm.13288 -
Gut Sep 2023Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as...
OBJECTIVE
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as syntenin1, as a novel targetable factor in promoting PDAC tumour progression. We also explored a therapeutic strategy for suppressing SDCBP expression.
DESIGN
We used samples from patients with PDAC, human organoid models, LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse models, and PDX mouse models. Immunostaining, colony formation assay, ethynyl-2-deoxyuridine incorporation assay, real-time cell analysis, cell apoptosis assay, automated cell tracking, invadopodia detection and gelatin degradation assays, coimmunoprecipitation, and pull-down assays were performed in this study.
RESULTS
The median overall survival and recurrence-free survival rates in the high-SDCBP group were significantly shorter than those in the low-SDCBP group. In vitro and in vivo studies have demonstrated that SDCBP promotes PDAC proliferation and metastasis. Mechanically, SDCBP inhibits CK1δ/ε-mediated YAP-S384/S387 phosphorylation, which further suppresses β-TrCP-mediated YAP1 ubiquitination and proteasome degradation by directly interacting with YAP1. SDCBP interacts with the TAD domain of YAP1, mainly through its PDZ1 domain. Preclinical KPC mouse cohorts demonstrated that zinc pyrithione (ZnPT) suppresses PDAC tumour progression by suppressing SDCBP.
CONCLUSIONS
SDCBP promotes the proliferation and metastasis of PDAC by preventing YAP1 from β-TrCP-mediated proteasomal degradation. Therefore, ZnPT could be a promising therapeutic strategy to inhibit PDAC progression by suppressing SDCBP.
Topics: Humans; Mice; Animals; beta-Transducin Repeat-Containing Proteins; Pancreatic Neoplasms; Pancreas; Carcinoma, Pancreatic Ductal; Cell Proliferation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Syntenins
PubMed: 36828627
DOI: 10.1136/gutjnl-2022-327492 -
Signal Transduction and Targeted Therapy Mar 2024NF-κB signaling has been discovered for nearly 40 years. Initially, NF-κB signaling was identified as a pivotal pathway in mediating inflammatory responses. However,... (Review)
Review
NF-κB signaling has been discovered for nearly 40 years. Initially, NF-κB signaling was identified as a pivotal pathway in mediating inflammatory responses. However, with extensive and in-depth investigations, researchers have discovered that its role can be expanded to a variety of signaling mechanisms, biological processes, human diseases, and treatment options. In this review, we first scrutinize the research process of NF-κB signaling, and summarize the composition, activation, and regulatory mechanism of NF-κB signaling. We investigate the interaction of NF-κB signaling with other important pathways, including PI3K/AKT, MAPK, JAK-STAT, TGF-β, Wnt, Notch, Hedgehog, and TLR signaling. The physiological and pathological states of NF-κB signaling, as well as its intricate involvement in inflammation, immune regulation, and tumor microenvironment, are also explicated. Additionally, we illustrate how NF-κB signaling is involved in a variety of human diseases, including cancers, inflammatory and autoimmune diseases, cardiovascular diseases, metabolic diseases, neurological diseases, and COVID-19. Further, we discuss the therapeutic approaches targeting NF-κB signaling, including IKK inhibitors, monoclonal antibodies, proteasome inhibitors, nuclear translocation inhibitors, DNA binding inhibitors, TKIs, non-coding RNAs, immunotherapy, and CAR-T. Finally, we provide an outlook for research in the field of NF-κB signaling. We hope to present a stereoscopic, comprehensive NF-κB signaling that will inform future research and clinical practice.
Topics: Humans; Immunotherapy; NF-kappa B; Phosphatidylinositol 3-Kinases; Signal Transduction
PubMed: 38433280
DOI: 10.1038/s41392-024-01757-9 -
Cell Aug 2023All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between...
All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.
Topics: Humans; Gene Expression; HEK293 Cells; HeLa Cells; Mutation; Signal Transduction; Transcription Factors; Ubiquitin-Protein Ligases
PubMed: 37478862
DOI: 10.1016/j.cell.2023.06.015 -
Blood Jul 2023Immunomodulatory agents (IMiDs) are a cornerstone of treatment for patients with multiple myeloma. IMiDs are used in therapeutic combinations at all stages of disease... (Review)
Review
Immunomodulatory agents (IMiDs) are a cornerstone of treatment for patients with multiple myeloma. IMiDs are used in therapeutic combinations at all stages of disease and are approved as a single-agent maintenance treatment after autologous stem cell transplantation. However, patients become resistant to ongoing therapy over time and inevitably relapse. It is only in the last decade that the mechanism of IMiD action has been elucidated; through binding to the cereblon component of the CRL4CRBN E3 ubiquitin ligase, a set of neosubstrates is designated for degradation by the proteasome. In myeloma cells, this includes the zinc-finger B-cell transcription factors Ikaros and Aiolos, which, in turn, lead to decreased levels of IRF4 and c-MYC and cell death. As our knowledge of IMiD mechanism of action has advanced, the ability to study resistance mechanisms has also developed. This review explores the existing work on IMiD resistance and proposes areas of future research that may advance our understanding and management of this common clinical condition.
Topics: Humans; Multiple Myeloma; Immunomodulating Agents; Hematopoietic Stem Cell Transplantation; Transplantation, Autologous; Neoplasm Recurrence, Local; Biology; Ubiquitin-Protein Ligases
PubMed: 36929172
DOI: 10.1182/blood.2023019637 -
Circulation Research Jan 2024Increasing evidence suggests that long noncoding RNAs play significant roles in vascular biology and disease development. One such long noncoding RNA, , has been...
BACKGROUND
Increasing evidence suggests that long noncoding RNAs play significant roles in vascular biology and disease development. One such long noncoding RNA, , has been implicated in the development of tumors. Nevertheless, the precise role of in cardiovascular diseases, particularly atherosclerosis, has not been thoroughly elucidated. Thus, the primary aim of this investigation is to assess the influence of on vascular inflammation and the initiation of atherosclerosis.
METHODS
We generated knockin and (Apolipoprotein E) knockout mice () and global and proteasome subunit-β type-9 () double knockout mice (). To explore the roles of and in atherosclerosis, we fed the mice with a Western diet for 12 weeks.
RESULTS
Long noncoding RNA is significantly elevated in human atherosclerotic plaques. Strikingly, mice exhibited increased atherosclerosis development, plaque vulnerability, and vascular inflammation compared with mice. Moreover, the levels of VCAM1 (vascular adhesion molecule 1) and ICAM1 (intracellular adhesion molecule 1) were significantly upregulated in atherosclerotic lesions and serum of mice. Consistently, in vitro gain- and loss-of-function studies demonstrated that induced monocyte/macrophage adhesion to endothelial cells and increased VCAM1 and ICAM1 levels in a PSMB9-dependent manner. Mechanistic studies revealed that induced transcription by recruiting the transcription factor NONO (non-POU domain-containing octamer-binding protein) and binding to the promoter. PSMB9 (proteasome subunit-β type-9) elevated VCAM1 and ICAM1 expression via the upregulation of ZEB1 (zinc finger E-box-binding homeobox 1). deficiency decreased atherosclerotic lesion size, plaque vulnerability, and vascular inflammation in mice in vivo. Importantly, endothelial overexpression of -increased atherosclerosis and vascular inflammation were attenuated by knockout.
CONCLUSIONS
promotes vascular inflammation and atherosclerosis via the NONOPSMB9/ZEB1 axis. Our findings support the development of new long noncoding RNA-based strategies to counteract atherosclerotic cardiovascular disease.
Topics: Animals; Humans; Mice; Apolipoproteins E; Atherosclerosis; Endothelial Cells; Inflammation; Mice, Inbred C57BL; Mice, Knockout; Plaque, Atherosclerotic; Proteasome Endopeptidase Complex; RNA, Long Noncoding
PubMed: 38084631
DOI: 10.1161/CIRCRESAHA.122.322360 -
The EMBO Journal Sep 2023Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic...
Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.
Topics: Humans; Amyotrophic Lateral Sclerosis; Frontotemporal Lobar Degeneration; DNA-Binding Proteins; Neurons; RNA
PubMed: 37431963
DOI: 10.15252/embj.2022111719 -
The Journal of Clinical Investigation Oct 2023The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses....
The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Posttranslational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study found that TLR priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilize the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation as well as hyperinflammation caused by viral infection. The results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection-induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3, revealing a priming target that modulates NLRP3-dependent immunopathology.
Topics: Animals; Humans; Mice; COVID-19; Inflammasomes; Inflammation; Mice, Inbred NOD; NLR Family, Pyrin Domain-Containing 3 Protein; SARS-CoV-2; Ubiquitin-Protein Ligases; Protein Processing, Post-Translational
PubMed: 37651190
DOI: 10.1172/JCI161935