Protein ubiquitination in ovarian cancer immunotherapy:The progress and therapeutic strategy

Ovarian cancer is a common cancer for females,and the incidence and mortality rates are on the rise.Many treatment strategies have been developed for ovarian cancer,including chemotherapy and immunotherapy,but they are often ineffective and prone to drug resistance.Protein ubiquitination is an important class of post-translation modifications that have been found to be associated with various human diseases and cancer development.Recent studies have revealed that protein ubiquitination is involved in the progression of ovarian cancer and plays an important role in the tumor immune process.Moreover,the com-bination of ubiquitinase/deubiquitinase inhibitors and cancer immunotherapy approaches can effectively reduce treatment resistance and improve treatment efficacy,which provides new ideas for cancer treatment.Herein,we review the role of protein ubiquitination in relation to ovarian cancer immunotherapy and recent advances in the use of ubiquitinase/deubiquitinase inhibitorsin combil ationwitncaner mmunotherapy.

Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy

Activation of nuclear factor erythroid 2-related factor 2(Nrf2)by Kelch-like ECH-associated protein 1(Keap1)alkylation plays a central role in anti-inflammatory therapy.However,activators of Nrf2 through alkylation of Keap1-Kelch domain have not been identified.Deoxynyboquinone(DNQ)is a natural small molecule discovered from marine actinomycetes.The current study was designed to investigate the anti-inflammatory effects and molecular mechanisms of DNQ via alkylation of Keap1.DNQ exhibited significant anti-inflammatory properties both in vitro and in vivo.The pharmacophore responsible for the anti-inflammatory properties of DNQ was determined to be theα,β-unsaturated amides moieties by a chemical reaction between DNQ and N-acetylcysteine.DNQ exerted anti-inflammatory effects through activation of Nrf2/ARE pathway.Keap1 was demonstrated to be the direct target of DNQ and bound with DNQ through conjugate addition reaction involving alkylation.The specific alkylation site of DNQ on Keap1 for Nrf2 activation was elucidated with a synthesized probe in conjunction with liquid chromatography-tandem mass spectrometry.DNQ triggered the ubiquitination and subsequent degradation of Keap1 by alkylation of the cysteine residue 489(Cys489)on Keap1-Kelch domain,ultimately enabling the activation of Nrf2.Our findings revealed that DNQ exhibited potent anti-inflammatory capacity throughα,β-unsaturated amides moieties active group which specifically activated Nrf2 signal pathway via alkylation/ubiquitination of Keap1-Kelch domain,suggesting the potential values of targeting Cys489 on Keap1-Kelch domain by DNQ-like small molecules in inflammatory therapies.

Hydralazine represses Fpn ubiquitination to rescue injured neurons via competitive binding to UBA52

A major impedance to neuronal regeneration after peripheral nerve injury (PNI) is the activation of various programmed cell death mechanisms in the dorsal root ganglion. Ferroptosis is a form of programmed cell death distinguished by imbalance in iron and thiol metabolism, leading to lethal lipid peroxidation. However, the molecular mechanisms of ferroptosis in the context of PNI and nerve regeneration remain unclear. Ferroportin (Fpn), the only known mammalian nonheme iron export protein, plays a pivotal part in inhibiting ferroptosis by maintaining intracellular iron homeostasis. Here, we explored in vitro and in vivo the involvement of Fpn in neuronal ferroptosis. We first delineated that reactive oxygen species at the injury site induces neuronal ferroptosis by increasing intracellular iron via accelerated UBA52-driven ubiquitination and degradation of Fpn, and stimulation of lipid peroxidation. Early administration of the potent arterial vasodilator, hydralazine (HYD), decreases the ubiquitination of Fpn after PNI by binding to UBA52, leading to suppression of neuronal cell death and significant acceleration of axon regeneration and motor function recovery. HYD targeting of ferroptosis is a promising strategy for clinical management of PNI.

Ubiquitination in osteosarcoma:unveiling the impact on cell biology and therapeutic strategies

Ubiquitination,a multifaceted post-translational modification,regulates protein function,degradation,and gene expression.The pivotal role of ubiquitination in the pathogenesis and progression of cancer,including colorectal,breast,and liver cancer,is well-established.Osteosarcoma,an aggressive bone tumor predominantly affecting adolescents,also exhibits dysregulation of the ubiquitination system,encompassing both ubiquitination and deubiquitination processes.This dysregulation is now recognized as a key driver of osteosarcoma development,progression,and chemoresistance.This review highlights recent progress in elucidating how ubiquitination modulates tumor behavior across signaling pathways.We then focus on the mechanisms by which ubiquitination influences osteosarcoma cell function.Finally,we discuss the potential for targeting the ubiquitin-proteasome system in osteosarcoma therapy.By unraveling the impact of ubiquitination on osteosarcoma cell physiology,we aim to facilitate the development of novel strategies for prognosis,staging,treatment,and overcoming chemoresistance.

Role of deubiquitinase JOSD2 in the pathogenesis of esophageal squamous cell carcinoma

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is a deadly malignancy with limited treatment options.Deubiquitinases(DUBs)have been confirmed to play a crucial role in the development of malignant tumors.JOSD2 is a DUB involved in con-trolling protein deubiquitination and influencing critical cellular processes in cancer.AIM To investigate the impact of JOSD2 on the progression of ESCC.METHODS Bioinformatic analyses were employed to explore the expression,prognosis,and enriched pathways associated with JOSD2 in ESCC.Lentiviral transduction was utilized to manipulate JOSD2 expression in ESCC cell lines(KYSE30 and RESULTS )Preliminary research indicated that JOSD2 was highly expressed in ESCC tissues,which was associated with poor prognosis.Further analysis demonstrated that JOSD2 was upregulated in ESCC cell lines compared to normal esophageal cells.JOSD2 knockdown inhibited ESCC cell activity,including proliferation and colony-forming ability.Moreover,JOSD2 knockdown decreased the drug resistance and migration of ESCC cells,while JOSD2 overexpression enhanced these phenotypes.In vivo xenograft assays further confirmed that JOSD2 promoted tumor proliferation and drug resistance in ESCC.Mechanistically,JOSD2 appears to activate the MAPK/ERK and PI3K/AKT signaling pathways.Mass spectrometry was used to identify crucial substrate proteins that interact with JOSD2,which identified the four primary proteins that bind to JOSD2,namely USP47,IGKV2D-29,HSP90AB1,and PRMT5.CONCLUSION JOSD2 plays a crucial role in enhancing the proliferation,migration,and drug resistance of ESCC,suggesting that JOSD2 is a potential therapeutic target in ESCC.

玉米Ubiquitin启动子的克隆及功能鉴定

根据Cornejo发表的Ubiquitin启动子的序列,设计引物从玉米自交系18红中克隆出该启动子,测序证明与发表的序列具有98%的同源性,并构建了带有该启动子和GUS基因的植物表达载体,将重组质粒导入农杆菌LBA4404,用农杆菌介导法转入烟草和小麦愈伤中,通过GUS染色反应,证明克隆的启动子在单子叶和双子叶植物中均有活性。

Ubiquitin B在宫颈癌细胞凋亡中的作用及机制的初步探讨

目的:研究Ubiquitin B的过度表达对宫颈癌细胞凋亡的影响,并初步探讨其机制。方法:脂质体转染Ubiquitin B至宫颈癌细胞系HeLa细胞后,real-time PCR检测细胞内Ubiquitin B mRNA的表达水平;流式细胞术和DNA Ladder法检测其凋亡;PI法检测细胞周期变化;Western blot法检测凋亡相关蛋白Smad4和Mcl-1的表达。结果:转染Ubiquitin B后,HeLa细胞中Ubiquitin B mRNA的表达水平在转染24h后明显高于它在对照组细胞中的表达。与对照组相比,转染了Ubiquitin B的HeLa细胞凋亡显著增加。但Ubiquitin B的过表达对HeLa细胞的周期无明显影响。Western blot分析表明,凋亡相关蛋白Smad4在转染Ubiquitin B48h时表达增强,而Mcl-1表达明显减弱。结论:UbiquitinB可促进宫颈癌细胞系HeLa细胞凋亡,其机制可能是Ubiquitin B通过增强表达凋亡促进蛋白Smad4和降解凋亡抑制蛋白Mcl-1而促进了HeLa细胞的凋亡。

Fanconi Anemia and Ubiquitination

Fanconi anemia （FA） is a rare recessive hereditary disease characterized clinically by congenital defects, progressive bone-marrow failure, and cancer predisposition. Cells from FA patients exhibit hypersensitivity to DNA cross-linking agents, such as mitomycin C （MMC）. To date, at least 12 FA genes have been found deleted or mutated in FA cells, and 10 FA gene products form a core complex involved in FA/BRCA2 DNA repair pathway-FA pathway. The ubiquitin E3 ligase FANCL, an important factor of FA core complex, co-functions with a new ubiquitin conjugating enzyme UBE2T to catalyze the monoubiquitination of FANCD2. FANCD2-Ub binds BRCA2 to form a new complex located in chromatin foci and then take part in DNA repair process. The deubiquitylating enzyme USP1 removes the mono-ubiquitin from FANCD2-Ub following completion of the repair process, then restores the blocked cell cycle to normal order by shutting off the FA pathway. In a word, the FANCD2 activity adjusted exquisitely by ubiquitination and/or deubiquitination in vivo may co-regulate the FA pathway involving in variant DNA repair pathway.

Ubiquitin基因介导下的PRRSV GP5基因免疫特性研究

目的:探讨泛素基因对GP5基因免疫的影响。泛素-蛋白酶体途径是一种高效蛋白降解途径,主要负责真核细胞内蛋白选择性降解。方法:本研究将ORF5 DNA片段克隆到含泛素(Ub)基因的表达载体pCMV-Ub和pCMV载体,构建成重组质粒pCMV-Ub-GP5和pCMV-GP5。两种质粒DNA肌肉注射免疫BALb/c小鼠后,分别检测体液免疫反应和细胞免疫反应,比较GP5单基因和Ub-GP5融合基因DNA免疫所诱生免疫应答的强度。结果:二者均可诱生PRRSV ELJSA抗体和中和抗体,其抗体水平无明显差别,但Ub-GP5融合基因诱生的淋巴细胞反应和CTL反应明显高于GP5基因。结论:泛素基因可以促进GP5诱生细胞免疫反应。

Ubiquitin真核表达载体构建及在293T细胞中的表达

为构建泛素分子pcDNA3.1-Myc-ubiquitin真核表达载体,实现其在293T细胞中表达。采用人工合成ubiquitin基因序列并加入c-Myc标签序列及EcoRⅠ和BamHⅠ酶切位点的方法,克隆至pcDNA3.1真核表达载体中。重组表达质粒经PCR和测序鉴定正确后,脂质体法转染入293T细胞。Western blot和间接免疫荧光法分析重组质粒在细胞中的蛋白表达及定位情况。菌落PCR及测序等分析结果显示:成功构建了pcDNA3.1-Myc-ubiquitin真核表达载体。免疫荧光和Western-Blot结果显示:Myc-ubiquitin重组表达质粒在293T细胞中获得高效表达且蛋白定位于胞浆。由此可知,成功构建pcDNA3.1-Myc-ubiquitin融合表达载体并在293T细胞中高效表达,为课题组进一步探讨与泛素化修饰相关的neuritin的作用机制及功能奠定基础。

Wwp2 mediates Oct4 ubiquitination and its own auto-ubiquitination in a dosage-dependent manner

Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating en- dogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells （ECCs）, although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanisti- cally, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interest- ingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in con- trolling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosage- dependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.

Deubiquitinating enzyme regulation of the p53 pathway: A lesson from Otub1

Deubiquitination has emerged as an important mechanism of p53 regulation. A number of deubiquitinating enzymes(DUBs) from the ubiquitin-specific protease family have been shown to regulate the p53-MDM2-MDMX networks. We recently reported that Otub1, a DUB from the OTU-domain containing protease family, is a novel p53 regulator. Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity. Instead, it does so by inhibiting the MDM2 cognate ubiquitin-conjugating enzyme(E2) UbcH5. Otub1 also regulates other biological signaling through this non-canonical mechanism, suppression of E2, including the inhibition of DNA-damage-induced chromatin ubiquitination. Thus, Otub1 evolves as a unique DUB that mainly suppresses E2 to regulate substrates. Here we review the current progress made towards the understanding of the complex regulation of the p53 tumor suppressor pathway by DUBs, the biological function of Otub1 including its positive regulation of p53, and the mechanistic insights into how Otub1 suppresses E2.

Role of E3 ubiquitin ligases in lung cancer

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.Therefore,E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation,proliferation and apoptosis.E3 ubiquitin ligases are often found overexpressed in human cancers,including lung cancer,and their deregulation has been shown to contribute to cancer development.However,the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting.In this review,we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer.Furthermore,we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets.By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis,we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Identification of Ubiquitinated Proteins from Human Multiple Myeloma U266 Cells by Proteomics

Objective To identify ubiquitinated proteins from complex human multiple myeloma （MM） U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immunoprecipitation,LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites,which were identified by detecting signature peptides containing a GG-tag （114.1 Da） and an LRGG-tag （383.2 Da）.Results In total,52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified,respectively.Conclusion Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups.This suggested the involvement of many endogenous proteins in the ubiquitination in MM.

薄束轴索变性小鼠的轴索变性和Ubiquitin的相互关系

目的 探索ＧＡＤ小鼠中枢神经系统的轴索变性和Ｕｂｉｑｕｉｔｉｎ的相互关系。方法 用免疫组织化学 染色方法。结果 在ＧＡＤ小鼠中枢神经系统的轴索变性部位，出现了多数Ｕｂｉｑｕｉｔｉｎ阳性的点状构造物（ｄｏｔ－ｌｉｋｅ ｓｔｒｕｃｔｕｒｅｓ：ＤＳ）。这种ＤＳ从９周龄开始，首先在构成薄束路和脊髓小脑后路的上行纤维的远端区域－薄束核区和小 脑白质出现，逐渐按照延髓、颈髓、胸髓、腰髓的顺序，向细胞体方向逆行性地进展。１８周龄时波及到锥体路，３２周 龄时进一步波及到丘脑和嗅觉、视觉、听觉传导路。结论 Ｕｂｉｑｕｉｔｉｎ阳性的ＤＳ作为病理构造物，是伴随着ＧＡＤ 小 鼠中枢神经系统的轴索变性而出现特征性所见。根据其分布及数量可了解轴索变性的部位、程度及进展过程。

肾消康对糖尿病大鼠肾组织基因ubiquitin mRNA表达的影响

目的观察中药复方肾消康对糖尿病肾病大鼠的防治作用及对肾脏基因表达的影响,探讨DN的发病机制,揭示中药复方肾消康防治DN的作用机理,筛选药效作用靶点,为临床推广应用提供科学依据。方法采用链脲佐菌素(STZ)加高热量饮食建立糖尿病大鼠肾脏损伤模型。运用放免、生化、光镜、电镜及美国Affymetrix公司的基因表达谱芯片、RT-PCR等先进检测手段和方法,观察多项指标,通过聚类分析寻找和DN有重要相关性的基因,并进一步采用实时荧光定量RT-PCR法做验证实验研究。结果泛素mRNA(ubiquitin mRNA)在糖尿病大鼠肾脏表达上调,肾消康治疗组表达下调。结论应用Affymetrix Rat2302.0基因表达谱芯片检测糖尿病大鼠肾脏基因的表达,ubiquitin mRNA基因是筛选出的肾脏差异表达基因和药效靶点之一。糖尿病状态下,ubiquitin mRNA基因表达上调,与糖尿病肾脏损伤有重要相关性,而经肾消康治疗后大鼠肾脏组织中ubiquitinm RNA表达下调。肾消康对ubiquitin mRNA基因表达具有良性调节作用。

Ubiquitin基因介导下的PRRSV GP5基因免疫特性研究

泛素—蛋白酶体途径是一种高效蛋白降解途径,主要负责真核细胞内蛋白选择性降解。GP5囊膜糖蛋白是PRRSV的主要结构蛋白。为了评价泛素基因对GP5基因免疫的影响,本研究将ORF5 DNA片段克隆到含泛素(Ub)基因的表达载体pCMV-Ub和pcMV载体,构建成重组质粒pCMV-Ub-GP5和pCMV-GP5。两种质粒DNA肌肉注射免疫BALb/c小鼠后,分别检测体液免疫反应和特异的细胞免疫反应,比较单基因和融合基因DNA免疫所诱生免疫应答的强度。结果表明二者均可诱生抗体的效率无明显差别,但泛素的融合使得针对GP5的特异性细胞免疫反应明显增强。

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

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.