Ubiquitin Research Articles

Ubiquitin forms conventional decorated micelle structures with sodium dodecyl sulfate at saturation

The anionic surfactant sodium dodecyl sulfate (SDS) interacts strongly with most globular proteins and denatures and unfolds them. While scattering studies using X-rays and neutrons have shown that this denaturation generally leads to protein-decorated SDS micelles, a different SDS-decorated polypeptide model has recently been suggested for complexes between SDS and Ubiquitin (UBI), in which individual SDS molecules are distributed on a partially stretched protein. To resolve this apparent discrepancy, we have investigated the SDS-UBI system by a number of complementary techniques. Small-angle X-ray scattering (SAXS) provides the overall structure of the SDS-UBI complexes, Tyr fluorescence and circular dichroism follow changes in tertiary and secondary structure, and isothermal titration calorimetry determines the stoichiometries of complexes and the amount of free SDS as a function of [SDS]. At low [SDS], UBI preserves its folded structure but dimerizes to a small extent. At 4 SDS per UBI, a complex is formed with two UBI and a small shared SDS cluster with 8 SDS molecules. In these complexes UBI preserves most of its native fold. At 10–12 SDS per UBI, which remains below the critical micelle concentration under our conditions, UBI-covered SDS micelles form with four UBIs around a core of 40 SDSs. This implies a protein-assisted micellization and an associated unfolding of UBI involving a change from mainly β-strands to mainly α-helical secondary structure. As [SDS] is increased, the complex gradually changes so that finally only one UBI covers one micelle with a similar number of SDS molecules at SDS saturation. Thus, we conclude that SDS unfolds UBI by mechanisms very similar to those observed for other globular proteins, leading to a protein-decorated SDS micelle rather than an SDS-decorated unfolded polypeptide chain.

Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer

Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.

English

Bletilla striata (Thunb.) Reihb.f., a traditional Chinese herbal medicine, has attracted increasing attention because of its wide range of applicability to the medical field and chemical industry. B. striata has been identified to be particularly sensitive to high temperatures. Thus, the study of temperature stress on gene transcription is of interest in the field. Use of reliable reference genes is essential for qRT-PCR analysis of genes. However, little information regarding suitable reference genes for the genus Bletilla has been published. In this study, the sequences of seven potential reference genes in B. striata were obtained via a homology cloning strategy. These genes were as follows: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), α-tubulin (TUA), β-tubulin (TUB), ubiquitin (UBI), and NAC domain protein (NAC). We then evaluated the stability levels of these transcripts in different tissues (root, tuber, and leaf) exposed to high temperature using three conventional software and comprehensive RefFinder algorithms. The results indicated that 18S and TUB were the best internal control genes among different periods of heat treatment and that a combination of 18S and UBI was the best in different tissues. Altogether, 18S and UBI were identified to be the best reference genes for all the samples, while NAC and TUA were the least stable reference genes. The results will be useful for studies on target gene expression in plants of the Bletilla genus. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.

Simultaneous measurement of 1HC/N-R2's for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins.

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand-protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

The influence of surface waters on the bioavailability and toxicity of copper oxide nanoparticles to freshwater mussels

The increased commercial use of copper oxide nanoparticles (nCuO) led to the release of nanoparticles in wastewaters potentially harming the aquatic biota. The purpose of this study was to determine the toxic action of nCuO and dissolved Cu (II) to Dreissena bugensis freshwater mussels placed in 4 types of surface waters: aquarium, green (high conductivity), brown (high organic carbon) and 10 % municipal effluent (high conductivity and anthropogenic source of organic carbon). Mussels were exposed to 50 µg/L of nCuO or Cu (II) for 96 h at 15 °C in the above waters. The results revealed that the total Cu loadings were higher in mussels placed in organic-rich waters (brown and effluent) and exposed to either forms of Cu. Tissue Cu contents were correlated with air-time survival, lipid peroxidation, protein-ubiquitin levels and DNA strand breaks. Both surface water types and Cu forms influenced Zn (II) mobilization, glutathione S-transferase activity and protein turnover (ubiquitin binding). Based on the surface water properties, Cu (II) was more influenced by the levels and origin of the organic carbon content while nCuO was more influenced by the total suspended solids. In conclusion the toxicity of nCuO could be influenced by surface waters properties expecially when similar physiological targets are impacts by these treatments.

Selection and optimization of reference genes for RT-qPCR normalization: A case study in Solanum lycopersicum exposed to UV-B

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.

The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.

The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.

RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila.

Drosophila melanogaster utilizes innate immune response to defend against exogenous pathogens. The molecular regulation mechanism of the process is evolutionarily conserved. Research of the regulatory mechanisms of Drosophila innate immunity is greatly significant for understanding the modulation of the human innate immunity and the pathogenesis of related diseases. To explore novel regulators in the STING-dependent innate immune response in Drosophila, we utilized the double-stranded RNA-mediated gene expression silencing technique and the dual-luciferase reporter system in knockdown experiments on 9 genes encoding the ubiquitin ligase such as echinus (CG2904), usp16 (CG4165), smurf (CG4943), pellino (CG5212), usp47 (CG5486), diap2 (CG8293), dtraf2 (CG10961), roquin (CG16807) and usp10 (CG32479) in the S2 cells in vitro. The results suggested a negative correlation between CG16807 (roquin) and the STING signaling pathway. Further studies showed that over-expression of roquin in S2 cells significantly inhibited STING innate immune signaling. Meanwhile, Listeria infection experiments showed that knocking down of roquin markedly elevated the expression levels of anti-microbial peptides and inhibited the proliferation of Listeria, thus increasing the survival rates post pathogenic infection. Taken together, our results suggested that the RNA-binding protein Roquin negatively regulates the STING-dependent innate immune response in Drosophila. In view of the high correlation between Drosophila genes and human genes, this study provides a theoretical basis for further development of treatments for STING-related innate immune diseases in humans.

Mutations in two ERAD E3 ubiquitin ligase enzymes reduce spontaneous reversal frequency in Caenorhabditis elegans.

Mutations in two ERAD E3 ubiquitin ligase enzymes reduce spontaneous reversal frequency in Caenorhabditis elegans.

Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

Extracellular ubiquitin protects cardiomyocytes during ischemia/hypoxia by inhibiting mitochondrial apoptosis pathway through CXCR4

AimAcute myocardial infarction (AMI) is one of the deadliest diseases worldwide. The search for countermeasures to reduce cardiomyocytes death in the infarcted area has always been the focus of research. Ubiquitin (UB) is a small polypeptide mainly involved in proteasome-mediated protein degradation in cells, whereas extracellular UB in body fluids can also function through its receptor CXC chemokine receptor type 4 (CXCR4). This study aimed to explore the functional roles of extracellular UB in cardiomyocytes during ischemia/hypoxia (I/H). MethodsH9C2 cells were subjected to I/H treatment and cell injury was evaluated by cell viability, morphology changes and apoptosis rate. UB expression and levels of ubiquitinated proteins after I/H injury were measured. The effects of extracellular UB on I/H-induced cardiomyocytes apoptosis and the possible underlying mechanisms were studied. ResultsI/H injury induced the decrease of cell viability as well as enhanced impaired cell morphology and apoptosis rate in H9C2 cells. Levels of UB mRNA and ubiquitinated proteins were significantly up-regulated after I/H treatment, whereas the concentration of extracellular UB in the conditioned media did not show significant change and the intracellular mono-UB levels in cells were down-regulated. Extracellular UB treatment protected cardiomyocytes from I/H injury by inhibiting the overactivation of mitochondria-dependent apoptosis pathway and up-regulating autophagy level. Inhibition of CXCR4 receptor using AMD3100 abolished cardioprotective effects of extracellular UB. ConclusionThe up-regulation of UB was suggested to be an adaptive response to resist I/H-induced cardiomyocytes apoptosis, and additional extracellular UB treatment might serve as a new potential therapeutic drug for AMI.

Cardioprotective Potential of Exogenous Ubiquitin.

Ischemic heart disease (IHD) accounts for the majority of heart disease-related deaths worldwide. Ubiquitin (UB), found in all eukaryotic cells, is a highly conserved low molecular weight (~8.5kDa) protein. A well-known intracellular function of UB is to regulate protein turnover via the UB-proteasome system. UB is a normal constituent of plasma, and elevated levels of UB are observed in the serum of patients under a variety of pathological conditions. Recent studies provide evidence for cardioprotective potential of exogenous UB in the remodeling process of the heart in IHD, including effects on cardiac myocyte apoptosis, inflammatory response, and reorganization of the vasculature and extracellular matrix. This review summarizes functions of UB with an emphasis on the role of exogenous UB in myocardial remodeling in IHD.

How ubiquitin determines the fate of our proteins

How ubiquitin determines the fate of our proteins

Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.

Serum Biomarkers of Cerebral Insult in Neonates with Hypoxic-ischemic Encephalopathy

Background: Neonatal hypoxic-ischemic encephalopathy (NHIE) is one of the most common causes of fatality albeit the effort spent on the management. We aimed to measure the serum biomarkers of cerebral insult in neonates with hypoxic-ischemic encephalopathy and its correlation with treatment by erythropoietin (Epo). Methods: Serum biomarkers for brain injury were collected from neonates with NHIE at day zero and day five after affection. These biomarkers included brain-related proteins which included; ubiquitin carboxy-terminal hydrolase-L1 (UCH- L1), neuron enolase, Tau, and S100B. Furthermore, the study included different cytokines that can be traced in brain injury including; interleukins (ILs) 1b, 6, 8, 10, 12P70, and 13 in addition to tumor necrosis factor-alpha (NF-a), gamma interferon (IFN-g), monocyte chemoattractant protein-1, and brain-derived neurotrophic factor (BDNF). The brain injury was assessed by MRI after one year. Results: Among the included neonates, brain injury was associated with elevated baseline levels of ILs)1b, 6, 8, 10, and 13, IFN-g, TNF-a, Tau, UCH-L1, and S100B. Furthermore, elevated serum levels of Tau at the baseline and decreased level of BDNF at day five were correlated with a bad prognosis in one year. No correlation detected between Epo treatment and these biomarkers. Conclusions: The increased serum levels of the tested biomarkers at day zero were associated with brain insults in newborns with NHIE. Furthermore, BDNF and Tau were associated with worse prognosis, and there was no correlation detected between Epo treatment and these biomarkers.

Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan (Carya illinoinensis)

This study proposed the combination of PR26S and PP1 as a good choice for RT-qPCR normalization in pecan under abiotic stress, developing kernels, grafting, and various tissues. Reference gene selection is an essential pre-requisite to generate reliable results in real-time quantitative polymerase chain reaction (RT-qPCR) analysis. However, studies regarding systematic validation of suitable reference genes in pecan are still lacking. In this study, 17 candidate reference genes were selected and evaluated for their expression stabilities in pecan under various experimental conditions, including various tissues, developing kernels, grafting, and two plant tissues (leaves and roots) subjected to three abiotic stresses (salt, drought, and Zn deficiency). The stability of the candidate genes was assessed by geNorm, NormFinder, and BestKeeper, and their outputs were integrated to obtain a final comprehensive rank of stability based on the geometric mean. The results indicated that samples under different experimental conditions possessed their own best reference genes, and using two reference genes for RT-qPCR normalization was recommended for the tested experiments. Overall, the combination of 26S protease regulatory subunit 7A (PR26S) and serine/threonine-protein phosphatase-1 (PP1) was recognized as a good choice for RT-qPCR normalization in pecan across all the treatments. More importantly, the widely used alpha-tubulin (α-TUB), ubiquitin (UBQ), and actin (ACT) genes were not the best suitable reference genes in most of our experiments. Our results will be helpful for future gene-expression studies in pecan.

Ubiquitin: there's no quitting

Ubiquitin: there's no quitting

Role of Deubiquitinases in Human Cancers: Potential Targeted Therapy.

Deubiquitinases (DUBs) are involved in various cellular functions. They deconjugate ubiquitin (UBQ) from ubiquitylated substrates to regulate their activity and stability. Studies on the roles of deubiquitylation have been conducted in various cancers to identify the carcinogenic roles of DUBs. In this review, we evaluate the biological roles of DUBs in cancer, including proliferation, cell cycle control, apoptosis, the DNA damage response, tumor suppression, oncogenesis, and metastasis. This review mainly focuses on the regulation of different downstream effectors and pathways via biochemical regulation and posttranslational modifications. We summarize the relationship between DUBs and human cancers and discuss the potential of DUBs as therapeutic targets for cancer treatment. This review also provides basic knowledge of DUBs in the development of cancers and highlights the importance of DUBs in cancer biology.

Ubiquitin receptor protein complex

ubiquitin receptor protein complex