Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation

The ubiquitin system is crucial for the development and fitness of higher plants.De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L.(exposed to light for 1, 6, or 12 h)achieved through immunoprecipitation-based high-resolution mass spectrometry(MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites(fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones.Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.

Roles of histone ubiquitylation in DNA damage signaling

Histone ubiquitylation has emerged as an important chromatin modification associated with DNA damage signaling and repair pathways.These histone marks,laid down by E3 ubiquitin ligases that include RNF8 and RNF168,decorate chromatin domains surrounding DNA double-strand breaks(DSBs).Recent work implicated ubiquitylated histones in orchestrating cell cycle checkpoints,DNA repair and gene transcription.Here we summarize recent advances that contribute to our current knowledge of the highly dynamic nature of DSB-associated histone ubiquitylation,and discuss major challenges ahead in understanding the versatility of ubiquitin conjugation in maintaining genome stability.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke

Stroke is the second leading cause of death worldwide,and oxidative stress plays a crucial role.Celastrol exhibits strong antioxidant properties in several diseases;however,whether it can affect oxidation in cerebral ischemic-reperfusion injury(CIRI)remains unclear.This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms.Here,we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2(Nrf2).Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry,we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4(Nedd4)and then released Nrf2 from Nedd4 in astrocytes.Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI,which was significantly blocked by celastrol.Furthermore,by inhibiting oxidative stress and astrocyte activation,celastrol effectively rescued neurons from axon damage and apoptosis.Our study uncovered Nedd4 as a direct target of celastrol,and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction.Vascular smooth muscle cells(VSMCs),the main components of atherosclerotic plaque,switch from contractile to synthetic phenotypes during atherogenesis.Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis,and it can be reversely regulated by deubiquitinases.However,the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated.In this study,RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases,which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch.Further in vivo studies using Apoe−/−mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype.Moreover,VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro.Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation,adhesion,and proliferation.Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated.Mechanistically,we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβwith its catalytic triad,thereby reducing the K48-linked ubiquitylation of PDGFRβ.Inhibiting OTUB1 in VSMCs could promote PDGFRβdegradation via the ubiquitin–proteasome pathway,so it was beneficial in preventing VSMCs’phenotype switch.These findings revealed that knocking down OTUB1 ameliorated VSMCs’phenotype switch and atherosclerosis progression,indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Chemical tools for E3 ubiquitin ligase study

E3 ubiquitin ligases catalyze the final step of ubiquitylation,a crucial post-translational modification involved in almost every process in eukaryotic cells.E3 ubiquitin ligases are key regulators of cellular events,and the investigation into their functions and functioning mechanisms are research areas with great importance.Synthetic or semi-synthetic tools have greatly facilitated the research about the enzyme activity,distribution in different physiological events,and catalytic mechanism of E3 ubiquitin ligase.In this review,we summarize the development of chemical tools for E3 ubiquitin ligases with an emphasis on the synthetic routes.We show the utility of these chemical tools by briefly discussing their applications in biological research.

Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism

The obstruction of post-insulin receptor signaling is the main mechanism of insulin-resistant diabetes.Progestin and adipoQ receptor 3(PAQR3),a key regulator of inflammation and metabolism,can negatively regulate the PI3 K/AKT signaling pathway.Here,we report that gentiopicroside(GPS),the main bioactive secoiridoid glycoside of Gentiana manshurica Kitagawa,decreased lipid synthesis and increased glucose utilization in palmitic acid(PA) treated HepG2 cells.Additionally,GPS improved glycolipid metabolism in streptozotocin(STZ) treated high-fat diet(HFD)-induced diabetic mice.Our findings revealed that GPS promoted the activation of the PI3 K/AKT axis by facilitating DNA-binding protein 2(DDB2)-mediated PAQR3 ubiquitinated degradation.Moreover,results of surface plasmon resonance(SPR),microscale thermophoresis(MST) and thermal shift assay(TSA) indicated that GPS directly binds to PAQR3.Results of molecular docking and cellular thermal shift assay(CETSA) revealed that GPS directly bound to the amino acids of the PAQR3 NH2-terminus including Leu40,Asp42,Glu69,Tyr125 and Ser129,and spatially inhibited the interaction between PAQR3 and the PI3 K catalytic subunit(P110α) to restore the PI3 K/AKT signaling pathway.In summary,our study identified GPS,which inhibits PAQR3 expression and directly targets PAQR3 to restore insulin signaling pathway,as a potential drug candidate for the treatment of diabetes.

A non-metabolic function of hexokinase 2 in small cell lung cancer:promotes cancer cell stemness by increasing USP11-mediated CD133 stability

Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor growth in mice.In addition,HK2 expression was positively correlated with CD133 expression in human SCLC specimens,and their expression levels were associated with poor prognosis of SCLC patients.Conclusions:These results revealed a critical non-metabolic function of HK2 in promotion of cancer cell stemness.Our findings provided new insights into the multifaceted roles of HK2 in tumor development.

First small-molecule PROTACs for G protein-coupled receptors:inducing α1A-adrenergic receptor degradation

Proteolysis targeting chimeras(PROTACs)are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitinproteasome system(UPS),thereby selectively reducing the target protein level by the ubiquitinproteasome pathway.Nowadays,small-molecule PROTACs are gaining popularity as tools to desrade pathogenic protein.Herein,we present the first small-molecule PROTACs that can induce the alA-adrenergic receptor(α1 A-AR)degradation,which is also the first small-molecule PROTACs for G proteincoupled receptors(GPCRs)to our knowledge.These degradation inducers were developed through conjugation of knownα1-adrenergic receptors(α1-ARs)inhibitor prazosin and cereblon(CRBN)ligand pomalidomide through the different linkers.The representative compound 9 c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression,which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.

Deregulation of tumor suppressive ASXL1−PTEN/AKT axis in myeloid malignancies

Mutations of epigenetic regulators are pervasive in human tumors.ASXL1 is frequently mutated in myeloid malignancies.We previously found that ASXL1 forms together with BAP1 a complex that can deubiquitinylate mono-ubiquitinylated lysine 119 on histone H2A(H2AK119ub1),a Polycomb repressive mark.However,a complete mechanistic understanding of ASXL1 in transcriptional regulation and tumor suppression remains to be defined.Here,we find that depletion of Asxl1 confers murine 32D cells to IL3-independent growth at least partly due to sustained activation of PI3K/AKT signaling.Consistently,Asxl1 is critical for the transcriptional activation of Pten,a key negative regulator of AKT activity.Then we confirm that Asxl1 is specifically enriched and required for H2AK119 deubiquitylation at the Pten promoter.Interestingly,ASXL1 and PTEN expression levels are positively correlated in human blood cells and ASXL1 mutations are associated with lower expression levels of PTEN in human myeloid malignancies.Furthermore,malignant cells with ASXL1 downregulation or mutations exhibit higher sensitivity to the AKT inhibitor MK2206.Collectively,this study has linked the PTEN/AKT signaling axis to deregulated epigenetic changes in myeloid malignancies.It also provides a rationale for mechanism-based therapy for patients with ASXL1 mutations.