Establishment of a prognosis predictive model for liver cancer based on expression of genes involved in the ubiquitin-proteasome pathway

BACKGROUND The ubiquitin-proteasome pathway(UPP)has been proven to play important roles in cancer.AIM To investigate the prognostic significance of genes involved in the UPP and develop a predictive model for liver cancer based on the expression of these genes.METHODS In this study,UPP-related E1,E2,E3,deubiquitylating enzyme,and proteasome gene sets were obtained from the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,aiming to screen the prognostic genes using univariate and multivariate regression analysis and develop a prognosis predictive model based RESULTS Five genes(including autophagy related 10,proteasome 20S subunit alpha 8,proteasome 20S subunit beta 2,ubiquitin specific peptidase 17 like family member 2,and ubiquitin specific peptidase 8)were proven significantly correlated with prognosis and used to develop a prognosis predictive model for liver cancer.Among training,validation,and Gene Expression Omnibus sets,the overall survival differed significantly between the high-risk and low-risk groups.The expression of the five genes was significantly associated with immunocyte infiltration,tumor stage,and postoperative recurrence.A total of 111 differentially expressed genes(DEGs)were identified between the high-risk and low-risk groups and they were enriched in 20 and 5 gene ontology and KEGG pathways.Cell division cycle 20,Kelch repeat and BTB domain containing 11,and DDB1 and CUL4 associated factor 4 like 2 were the DEGs in the E3 gene set that correlated with survival.CONCLUSION We have constructed a prognosis predictive model in patients with liver cancer,which contains five genes that associate with immunocyte infiltration,tumor stage,and postoperative recurrence.

Progress of ubiquitin-proteasome system in the pathophysiology of heart failure and the intervention of traditional Chinese medicine

Heart failure(HF)represents one of the leading causes of morbidity and mortality in the modern world,which threatened approximately 1%~2%of adults’lives.HF is the end stage of multiple cardiovascular diseases characterized by cardiac hypertrophy and myocardial remodeling,and the pathophysiological processes of which include oxidative stress,endoplasmic reticulum homeostasis,apoptosis,autophagy,energy metabolism disorder,etc.The regulation of protein homeostasis intrinsically interrelates the above pathophysiological processes.Therefore,it is imperative to elucidate the molecular mechanism from the perspective of protein homeostasis to find new therapeutic targets for HF treatment.The dynamic regulation and post-translational modification of protein synthesis and degradation play a vital role in response of living organisms to physiological changes.The ubiquitin-proteasome system(UPS),which degrades 70-90%of endogenous proteins,plays an integral part in the pathophysiological processes of HF.The UPS can regulate oxidative stress,endoplasmic reticulum homeostasis,apoptosis and autophagy of cardiomyocytes(CMs),energy metabolism,targeting degradation signals and structural proteins,thus modulating cardiac hypertrophy,fibrosis and remodeling,finally contributing to the occurrence and progression of HF.Thus,regulating UPS is a promising effective strategy to treat HF.Increasing evidence indicates that traditional Chinese medicine(TCM)targeting the UPS is potential to ameliorate HF.This review will summarize the current knowledge focusing on the underlying mechanism and the important research advances related to UPS in treating HF,and the traditional Chinese medicine targeting UPS.

Effects of inhibition of ubiquitin-proteasome pathway on human primary leukemic cells

Though there were a lot of reports about the totally different responses to the inhibition of ubiquitin-proteasome pathway in different kinds of cell lines, much less has been known about the responses in primary human leukemic cells. In this study, the effects of inhibition of ubiquitin-proteasome pathway on human bone marrow (BM) mononuclear cells (MNCs) obtained from 10 normal persons and 8 leukemia patients were examined. The results showed that the responses obviously varied individually. Among them, BM MNCs in 3 cases of leukemic patients were extremely sensitive, demonstrated by that >90% cells were induced to undergo apoptosis within 24 h, but MNCs in 10 cases of normal persons showed resistance to the inhibition and no apoptosis was observed. Furthermore, Western blots revealed that the Bcl-2 expression was relatively high in the sensitive primary leukemia cells, and especially the cleavage of 26 ku Bcl-2 into a 22 ku fragment occurred during the induction of apoptosis. In contrast, the Bcl-2 expression was either undetectable or detectable but no cleavage of that above was observed in the cells insensitive to the inhibition of the pathway (including BM MNCs in normal persons). Together with the observations on the leukemic cell lines, these findings suggested the correlation of the specific cleavage of Bcl-2 into a shortened fragment with the sensitivity of cells to the inhibition of ubiquitin-proteasome pathway, which provides clues to the further understanding of the mechanisms of that dramatically different responses existing in different kinds of cells to the inhibition of ubiquitin-proteasome pathway.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer’s disease by the high-content fluorescence imaging system

Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.

Myelinosome organelles in pathological retinas: ubiquitous presence and dual role in ocular proteostasis maintenance

The timely and efficient elimination of aberrant proteins and damaged organelles, formed in response to various genetic and environmental stressors, is a vital need for all cells of the body. Recent lines of evidence point out several non-classical strategies employed by ocular tissues to cope with aberrant constituents generated in the retina and in the retinal pigmented epithelium cells exposed to various stressors. Along with conventional strategies relying upon the intracellular degradation of aberrant constituents through ubiquitin-proteasome and/or lysosome-dependent autophagy proteolysis, two non-conventional mechanisms also contribute to proteostasis maintenance in ocular tissues. An exosome-mediated clearing and a myelinosome-driven secretion mechanism do not require intracellular degradation but provide the export of aberrant constituents and “waste proteins” outside of the cells. The current review is centered on the non-degradative myelinosome-driven secretion mechanism, which operates in the retina of transgenic Huntington’s disease R6/1 model mice. Myelinosome-driven secretion is supported by rare organelles myelinosomes that are detected not only in degenerative Huntington’s disease R6/1 retina but also in various pathological states of the retina and of the retinal pigmented epithelium. The intra-retinal traffic and inter-cellular exchange of myelinosomes was discussed in the context of a dual role of the myelinosome-driven secretion mechanism for proteostasis maintenance in different ocular compartments. Special focus was made on the interplay between degradative and non-degradative strategies in ocular pathophysiology, to delineate potential therapeutic approaches to counteract several vision diseases.

IGF-1 promotes the growth and metastasis of hepatocellular carcinoma via the inhibition of proteasome-mediated cathepsin B degradation

AIM: To investigate the molecular mechanisms of the high IGF-1 level linking diabetes and cancers, which is a risk factor.METHODS: We used cell growth, wound healing and transwell assay to evaluate the proliferation and metastasis ability of the hepatocellular carcinoma(HCC) cells. Western blot and reverse transcription polymerase chain reaction were used to assess a previously identified lysosomal protease, cathepsin B(CTSB) expression in the HCC cell lines. C57 BL/6J and KK-Ay diabetic mice are used to detect the growth and metastasis of HCC cells that were depleted with or without CTSB sh RNA in vivo. Statistical significance was determined by Student's t-test.RESULTS: IGF-1 promoted the growth and metastasis of the HCC cell lines via its ability to enhance CTSB expression in both a time-dependent and concentration-dependent manner. HCC cells grew much faster in diabetic KK-Ay mice than in C57 BL/6J mice. Additionally, more metastatic nodules were found in the lungs of KK-Ay mice than the lungs of C57 BL/6J mice. CTSB depletion protects against the tumorpromoting actions of IGF-1 in HCC cells, as well tumor growth and metastasis both in vitro and in vivo.IGF-1 did not change the m RNA levels of CTSB but prolonged the half-life of cathepsin B in Hepa 1-6 and H22 cells. Our results showed that IGF-1 promotes the growth and metastasis of the HCC cells most likely by hindering CTSB degradation mediated by the ubiquitinproteasome system(UPS), but not autophagy. Overexpression of proteasome activator 28, a family of activators of the 20 S proteasome, could not only restore IGF-1-inhibited UPS activity but also decrease IGF-1-induced CTSB accumulation.CONCLUSION: Our study demonstrates that IGF-1 promotes the growth and metastasis of hepatocellular carcinoma by inhibition of proteasome-mediated CTSB degradation.

New insights into estrogenic regulation of O^6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells： Co-degradation of ER-α and MGMT proteins by fulvestrant or O^6-benzylguanine indicates fresh avenues for therapy

Endocrine therapy using estrogen receptor-u （ER-α） antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O^6-methylguanine DNA methyltransferase （MGMT）, a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines （MCF-7, T47D） and ER- negative cell lines （MDAMB- 468, MDAMB-231）, and established inhibitors of ER-α and MGMT, namely, ICI-182,780 （Faslodex） and O^6- benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements （EREs） and two antioxidant-responsive elements （AREs）. MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 （nuclear factor-erythroid 2-related factor-2） increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this dragresistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O^6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O^6-benzylguanine also induced a specific loss of ER-a and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-a and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-a proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ER-negative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.

Anti-tumor Action and Clinical Application of Proteasome Inhibitor

Ubiquitin-proteasome pathway mediates the degradation of cell protein, and cell cycle, gene translation and expression, antigen presentation and inflammatory development. Proteasome inhibitor can inhibit growth and proliferation of tumor cell, induce apoptosis and reverse multipledrug resistance of tumor cell, increase the sensitivity of other chemotherapeutic drugs and radiotherapy, and is a novel class of potent anti-tumor agents.

Ubiquitin-conjugating enzyme involved in the immune response caused by pathogens invasion

Ubiquitin-proteasome pathway (UPP) is a significant way of protein degradation and modification in eukaryotic cell and involved in a complex series of intracellular processes. As a key component in UPP,?ubiquitin-conjugating enzyme (E2) plays an extremely important role in ubiquitin (Ub) transferring and substrate specific recognition. Abundant evidences have proved that UPP is involved in cells immune reaction caused by pathogens and the attendance of E2 has a significant effect on host cells and pathogen. This article presents an overview of the current research on E2s that is involved in immune response caused by viruses and bacteria.

Oxidative modification of the molecular chaperone family in a PC12 cell model of Parkinson's disease induced by Z-Ile-Glu(OtBu)-Ala-Leucinal

Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson＇s disease patients. In the present study, proteasome inhibitor Z-Ile-Glu（OtBu）-Ala-Leucinal （PSI） was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson＇s disease. Oxidatively modified proteins were identified to determine pathogenesis of Parkinson＇s disease. Results demonstrated that 24 hours of 10 IJmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson＇s disease： neuronal degeneration and eosinophilic inclusion formation in neurons. In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected： chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58, and heat shock protein 70. This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson＇s disease induced with PSI.

Anchanling reduces pathology in a lactacystin-induced Parkinson's disease model

A rat model of Parkinson＇s disease was induced by injecting lactacystin stereotaxically into the left mesencephalic ventral tegmental area and substantia nigra pars compacta. After rats were intragastrically perfused with Anchanling, a Chinese medicine, mainly composed of magnolol, for 5 weeks, when compared with Parkinson’s disease model rats, tyrosine hydroxylase expression was increased, α-synuclein and ubiquitin expression was decreased, substantia nigra cell apoptosis was reduced, and apomorphine-induced rotational behavior was improved. Results suggested that Anchanling can ameliorate Parkinson＇s disease pathology possibly by enhancing degradation activity of the ubiquitin-proteasome system.

Differential Expression of Molecular Chaperones in PC12 Cells Treated with PSI

Parkinson＇s disease（PD） is a common neurodegenerative disorder whose primary pathology features are the degeneration of dopaminergic neurons in the substantia nigra pars compacta（SNc） and the presence of eosinophilic inclusions called Lewy body in the cytoplasm of the remained neurons. Growing evidence suggests that dysfunction of the ubiquitin-proteasome system（UPS） is involved in the etiopathogenesis of PD. In order to investigate the pathogenetic mechanism of ubiquitin-proteasome dysfunction in PD, 2D-differential gel electrophoresis（2D-DIGE） and MALDI-TOF Pro MS were used to determine the proteins, which were differentially expressed, in PC12 cells that had undergone a synthetic proteasomal inhibitor PSI（10 μmol/L） treatment for 24 h. Forty-six protein spots were differentially expressed in response to PSI administration, of which 34 were increased and 12 decreased. Six of these were identified as molecular charperones： endoplasmin precursor（GRP94）, heat shock protein 105（HSP105）, HSC-70-psl, glucose ruglated protein 75（GRP75）, glucose ruglated protein 58（GRP58） and heat shock 27000 protein l（HSP27）. The results suggest that the molecular chaperones play an important role in the PD model induced by proteasomal inhibitor.

Increased expression of the 20S proteasome in peripheral blood mononuclear cells of type 2 diabetic patients

Objective To investigate the dynamic expression of the 20S proteasome in peripheral blood mononuclear cells(PBMCs)of type 2 diabetic patients without vascular complications.Methods PBMCs were prepared from 30 type 2 diabetic patients and 30 nondiabetic controls.The general indexes including weight,height and blood pressure were recorded.Fasting plasma glucose,fasting plasma insulin and glycosylated hemoglobin were measured.The protein level of the 20S proteasome was measured by Western blotting.The mRNA expression levels of the 20S proteasome β1,β2 and β5 subunits were detected by real-time PCR.Results Compared with that in the nondiabetic controls,the protein level of the 20S proteasome was significantly increased in the diabetic patients and was positively associated with glycosylated hemoglobin.Conclusion Type 2 diabetic patients without vascular complications have an increased 20S proteasome expression,the significance of which needs to be explored by further study.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.

Regulation of chloroplast protein degradation

Chloroplasts are unique organelles that not only provide sites for photosynthesis and many metabolic processes,but also are sensitive to various environmental stresses.Chloroplast proteins are encoded by genes from both nuclear and chloroplast genomes.During chloroplast development and responses to stresses,the robust protein quality control systems are essential for regulation of protein homeostasis and the integrity of chloroplast proteome.In this review,we summarize the regulatory mechanisms of chloroplast protein degradation refer to protease system,ubiquitin-proteasome system,and the chloroplast autophagy.These mechanisms symbiotically play a vital role in chloroplast development and photosynthesis under both normal or stress conditions.

Large yellow croaker(Larimichthys crocea)mitofusin 2 inhibits type I IFN responses by degrading MAVS via enhanced K48‑linked ubiquitination

In mammals,mitofusin 2(MFN2)is involved in mitochondrial fusion,and suppresses the virus-induced RIG-I-like receptor(RLR)signaling pathway.However,little is known about the function of MFN2 in non-mammalian species.In the present study,we cloned an MFN2 ortholog(LcMFN2)in large yellow croaker(Larimichthys crocea).Phylogenetic analysis showed that MFN2 emerged after the divergence of amphioxus and vertebrates.The protein sequences of MFN2 were well conserved from fsh to mammals.LcMFN2 was expressed in all the tissues/organs examined at diferent levels,and its expression was upregulated in response to poly(I:C)stimulation.Overexpression of LcMFN2 inhibited MAVS-induced type I interferon(IFN)promoter activation and antiviral gene expression.In contrast,knockdown of endogenous LcMFN2 enhanced poly(I:C)induced production of type I IFNs.Additionally,LcMFN2 enhanced K48-linked polyubiquitination of MAVS,promoting its degradation.Also,overexpression of LcMFN2 impaired the cellular antiviral response,as evidenced by the increased expression of viral genes and more severe cytopathic efects(CPE)in cells infected with spring viremia of carp virus(SVCV).These results indicated that LcMFN2 inhibited type I IFN response by degrading MAVS,suggesting its negative regulatory role in cellular antiviral response.Therefore,our study sheds a new light on the regulatory mechanisms of the cellular antiviral response in teleosts.

Degradation of proteins by PROTACs and other strategies

Abnormal protein expression or activities are associated with many diseases,especially cancer.Therefore,down-regulating the proteins involved in cancer cell survival proved to be an effective strategy for cancer treatment—a number of drugs based on proteolysis-targeting chimaera(PROTAC)mechanism have demonstrated clinical efficacy.Recent progress in the PROTAC strategy includes identification of the structure of the first ternary eutectic complex,extra-terminal domain-4-PROTAC-VonHippel-Lindau(BRD4-PROTAC-VHL),and PROTAC ARV-110 has entered clinical trials for the treatment of prostate cancer in 2019.These discoveries strongly proved the value of the PROTAC strategy.In this review,we summarize recent meaningful research of PROTACs,including the molecular design and optimization strategy as well as clinical application of candidate molecules.We hope to provide useful insights for rational design of PROTACs.

Anticancer drug discovery by targeting cullin neddylation

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade.The bestknown substrates of neddylation are cullin family proteins,which are the core component of Cullin-RING E3 ubiquitin ligases(CRLs).Given that cullin neddylation is required for CRL activity,and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers,targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics.In the past decade,we have witnessed significant progress in the field of protein neddylation from preclinical target validation,to drug screening,then to the clinical trials of neddylation inhibitors.In this review,we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade,followed by a summary of all reported chemical inhibitors of neddylation enzymes.We then discussed the structure-based targeting of protein-protein interaction in neddylation cascade,and finally the available approaches for the discovery of new neddylation inhibitors.This review will provide a focused,up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.

A Bunyavirus-Inducible Ubiquitin Ligase Targets RNA Polymerase IV for Degradation during Viral Pathogenesis in Rice

The ubiquitin-proteasome system(UPS)is an important post-translational regulatory mechanism that controls many cellular functions in eukaryotes.Here,we show that stable expression of P3 protein encoded by Rice grassy stunt virus(RGSV),a negative-strand RNA virus in the Bunyavirales,causes developmental abnormities similar to the disease symptoms caused by RGSV,such as dwarfing and excess tillering,in transgenic rice plants.We found that both transgenic expression of P3 and RGSV infection induce ubiquitination and UPS-dependent degradation of rice NUCLEAR RNA POLYMERASE D1a(OsNRPD1a),one of two orthologs of the largest subunit of plant-specific RNA polymerase IV(Pol IV),which is required for RNA-directed DNA methylation(RdDM).Furthermore,we identified a P3-inducible U-box type E3 ubiquitin ligase,designated as P3-inducible protein 1(P3IP1),which interacts with OsNRPD1a and mediates its ubiquitination and UPS-dependent degradation in vitro and in vivo.Notably,both knockdown of OsNRPD1 and overexpression of P3IP1 in rice plants induced developmental phenotypes similar to RGSV disease symptomss.Taken together,our findings reveal a novel virulence mechanism whereby plant pathogens target host RNA Pol IV for UPS-dependent degradation to induce disease symptoms.Our study also identified an E3 ubiquitin ligase,which targets the RdDM compotent NRPD1 for UPS-mediated degradation in rice.