AtPPRT1, an E3 Ubiquitin Ligase, Enhances the Thermotolerance in Arabidopsis.
ABSTRACT: E3 ubiquitin ligase plays a vital role in the ubiquitin-mediated heat-related protein degradation pathway. Herein, we report that the expression of AtPPRT1, a C3HC4 zinc-finger ubiquitin E3 ligase gene, was induced by heat stress, and the ?-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity after basal and acquired thermotolerance. To further explore the function of AtPPRT1 in heat stress response (HSR), we used the atpprt1 mutant and AtPPRT1-overexpressing lines (OE2 and OE10) to expose in heat shock. In this study, the atpprt1 mutant had a lower germination and survival rate than those of Col-0 when suffered from the heat stress, whereas OEs enhanced basal and acquired thermotolerance in Arabidopsis seedlings. When compared to Col-0 and OEs, loss-of-function in AtPPRT1 resulted in lower chlorophyll retention and higher content of reactive oxygen species (ROS) after heat treatment. Moreover, the transcript levels of AtPPRT1 and several heat-related genes (AtZAT12, AtHSP21 and AtHSFA7a) were upregulated to greater extents in OEs and lower extents in atpprt1 compared to Col-0 after heat treated. Hence, we suggest that AtPPRT1 may act as a positive role in regulating the high temperature by mediating the degradation of unknown target proteins.
Project description:In plants, 14-3-3 proteins are recognized as mediators of signal transduction and function in both development and stress response. However, there are only a few preliminary functional researches in the C4 crop foxtail millet. Here, phylogenetic analysis categorized foxtail millet 14-3-3s (SiGRFs) into 10 discrete groups (Clusters I to X). Transcriptome and qPCR analyses showed that all the SiGRFs responded to at least one abiotic stress. All but one SiGRF-overexpressing (OE) Arabidopsis thaliana line (SiGRF1) exhibited insensitivity to abiotic stresses during seed germination and seedling growth. Compared with the Col-0 wild-type, SiGRF1-OEs had slightly lower germination rates and smaller leaves. However, flowering time of SiGRF1-OEs occurred earlier than that of Col-0 under high-salt stress. Interaction of SiGRF1 with a foxtail millet E3 ubiquitin-protein ligase (SiRNF1/2) indicates that the proteinase system might hydrolyze SiGRF1. Further investigation showed that SiGRF1 localized in the cytoplasm, and its gene was ubiquitously expressed in various tissues throughout various developmental stages. Additionally, flowering-related genes, WRKY71, FLOWERING LOCUS T, LEAFY, and FRUITFULL, in SiGRF1-OEs exhibited considerably higher expression levels than those in Col-0 under salinity-stressed conditions. Results suggest that SiGRF1 hastens flowering, thereby providing a means for foxtail millet to complete its life cycle and avoid further salt stress.
Project description:Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.
Project description:The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Project description:Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. For this study, a target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. In contrast to mock-infected cells, only PML-GFP-expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML-GFP when examined by fluorescence microscopy and FCM analysis. Our results suggest that it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture by FCM analysis.
Project description:The heat shock response is crucial for organisms against heat-damaged proteins and maintaining homeostasis at a high temperature. Heterologous expression of eukaryotic molecular chaperones protects Escherichia coli from heat stress. Here we report that expression of the plant E3 ligase BnTR1 significantly increases the thermotolerance of E. coli. Different from eukaryotic chaperones, BnTR1 expression induces the accumulation of heat shock factor ?32 and heat shock proteins. The active site of BnTR1 in E. coli is the zinc fingers of the RING domain, which interacts with DnaK resulting in stabilizing ?32. Our findings indicate the expression of BnTR1 confers thermoprotective effects on E. coli cells, and it may provide useful clues to engineer thermophilic bacterial strains.
Project description:The heat-shock response is an evolutionarily conserved cellular defense mechanism against environmental stresses, characterized by the rapid synthesis of heat-shock proteins (HSPs). HSP70, a highly inducible molecular chaperone, assists in refolding or clearance of damaged proteins, thereby having a central role in maintaining intracellular homeostasis and thermotolerance. To date, induction of HSP70 expression has been described extensively at the transcriptional level. However, post-translational regulation of HSP70, such as protein stability, is only partially understood. In this study, we investigated the role of OLA1 (Obg-like ATPase 1), a previously uncharacterized cytosolic ATPase, in regulating the turnover of HSP70. Downregulation of OLA1 in mammalian cells by either RNAi or targeted gene disruption results in reduced steady-state levels of HSP70, impaired HSP70 induction by heat, and functionally, increased cellular sensitivity to heat shock. Conversely, overexpression of OLA1 correlates with elevated HSP70 protein levels and improved thermal resistance. Protein-protein interaction assays demonstrated that binding of OLA1 to the HSP70 carboxyl terminus variable domain hinders the recruitment of CHIP (C-terminus of Hsp70-binding protein), an E3 ubiquitin ligase for HSP70, and thus prevents HSP70 from the CHIP-mediated ubiquitination. These findings suggest a novel molecular mechanism by which OLA1 stabilizes HSP70, leading to upregulation of HSP70 as well as increased survival during heat shock.
Project description:BACKGROUND:Heat stress is a severe environmental stress that affects plant growth and reduces yield. Bax inhibitor-1 (BI-1) is a cytoprotective protein that is involved in the response to biotic and abiotic stresses. The Arabidopsis (Arabidopsis thaliana) BI-1 mutants atbi1-1 and atbi1-2 are hypersensitive to heat stress, and AtBI-1 overexpression rescues thermotolerance deficiency in atbi1 plants. Nevertheless, the mechanism of BI-1 in plant thermotolerance is still unclear. RESULTS:We identified a wheat (Triticum aestivum L.) BI-1 gene, TaBI-1.1, which was highly upregulated in an RNA sequencing (RNA-seq) analysis of heat-treated wheat. The upregulation of TaBI-1.1 under heat stress was further demonstrated by real time quantitative PCR (qRT-PCR) and ?-glucuronidase (GUS) staining. Compared with the wild type Col-0, the atbi1-2 mutant is hypersensitive to heat stress, and constitutive expression of TaBI-1.1 in atbi1-2 (35S::TaBI-1.1/ atbi1-2) rescued the deficiency of atbi1-2 under heat stress. Furthermore, we identified TaFKBP62 as a TaBI-1.1-interacting protein that co-localized with TaBI-1.1 on the endoplasmic reticulum (ER) membrane and enhanced heat stress tolerance. Additionally, HSFA2, HSFB1, ROF1, HSP17.4B, HSP17.6A, HSP17.8, HSP70B, and HSP90.1 expression levels were suppressed in atbi1-2 plants under heat stress. In contrast, 35S::TaBI-1.1/atbi1-2 relieved the inhibitory effect of AtBI-1 loss of function. CONCLUSIONS:TaBI-1.1 interacted with TaFKBP62 and co-localized with TaFKBP62 on the ER membrane. Both TaBI-1.1 and AtBI-1 regulated the expression of heat-responsive genes and were conserved in plant thermotolerance.
Project description:NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1-related ubiquitin ligases.
Project description:Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear-cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock.
Project description:OBJECTIVE:This study aimed to investigate the effect of circular RNA itchy E3 ubiquitin protein ligase on cell proliferation and apoptosis and to explore its target micro-RNAs in prostate cancer cells. METHODS:Circular RNA itchy E3 ubiquitin protein ligase expression in human prostate cancer cells and normal prostate epithelial cells was determined by real time-quantitative polymerase chain reaction assay. Circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase(+) group and control overexpression plasmids group were transfected with PC-3 cells. Rescue experiment was performed by transfection of circular RNA itchy E3 ubiquitin protein ligase overexpression and micro-197 overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group) into PC-3 cells. Cell Counting Kit-8 and annexin V/propidium iodide assays were conducted to evaluate cell proliferation and apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic-related markers. RESULTS:Circular RNA itchy E3 ubiquitin protein ligase expression was decreased in DU 145, 22RV1, VCaP, and PC-3 cells compared to RWPE cells. In PC-3 cells, cell proliferation rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours and 72 hours. Cell apoptosis rate was elevated in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours, and Western blot showed the similar results. Micro RNA-197 but not micro RNA-31 or micro RNA-432 was the target micro-RNA of circular RNA itchy E3 ubiquitin protein ligase. In rescue experiments, cell proliferation rate was elevated, but apoptosis rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group compared to circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group, indicating that circular RNA itchy E3 ubiquitin protein ligase upregulation inhibited cell proliferation but promoted apoptosis through downregulating micro RNA-197. CONCLUSION:Circular RNA itchy E3 ubiquitin protein ligase upregulation suppresses cell proliferation but promotes apoptosis through targeting micro RNA-197 in prostate cancer. Our study may provide a new insight for the treatment of prostate cancer.