Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

Small ubiquitin-like modifier protein-specific protease 1 and prostate cancer

Small ubiquitin-like modifier protein （SUMO） modification is a highly dynamic process, catalyzed by SUMO- specific activating （El）, conjugating （E2） and ligating （E3） enzymes, and reversed by a family of SUMO-specific proteases （SENPs）. There are six members of the human SENP family, and each SENP has different cellular locations and substrate specificities. However, the precise roles of SENPs in cellular processes have not been elucidated to date. This brief review will focus on recent advances pertaining to the identified targets of SENP 1 and its potential role in prostate cancer.

A dicistrovirus increases pupal mortality in Spodoptera frugiperda by suppressing protease activity and inhibiting larval diet consumption

Understanding interactions between viruses and their hosts is conducive to enabling better application of viruses as biocontrol agents.Certain viruses carried by parasitic wasps enhance the parasitic efficiency of wasp-larvae by protecting them against the immune system of their Lepidopteran host.However,the relationship between prey pests and viruses found in predatory natural enemies remains unclear.Herein,we report the interaction between Arma chinensis virus-1(AcV-1),originally isolated from a predatory natural enemy,Arma chinensis(Hemiptera:Pentatomidae),and one of its prey species,Spodoptera frugiperda(Lepidoptera:Noctuidae).The results showed that the AcV-1 virus appeared harmful to the novel host S.frugiperda by inhibiting larval diet consumption and increasing pupal mortality.Meanwhile,sequencing data indicated that the virus altered the gene expression profiles of S.frugiperda.KEGG analysis showed that the proteasome and phagosome pathways related to protein degradation and immune response were significantly enriched.Although the expression levels of digestive enzyme genes did not change significantly,the total protease activity of AcV-1 virus-positive individuals was significantly decreased,suggesting that the virus inhibited diet consumption of S.frugiperda via the down-regulation of digestive enzyme activities.These results indicate that a virus initially isolated in a predatory natural enemy can decrease the fitness of its prey species.The virus was found to impact the host proteasome and phagosome pathways related to protein degradation and immunity,providing a potential mechanism to enhance controlling efficiency.

Serine protease HtrA1 expression in human hepatocellular carcinoma

BACKGROUND: HtrA1, a serine protease, is down-regulated in various human solid tumors. Overexpression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, suggesting its possible role as a tumor suppressor. METHODS: Immunohistochemistry was used to determine the expression of HtrA1 in 50 hepatocellular carcinoma specimens and adjacent liver tissues. The correlation between the expression of HtrA1 and the clinico-pathologic data were analyzed. RESULTS: The levels of HtrA1 were lower in tumor tissues than in their adjacent liver tissues. Moreover, an inverse relationship was found between HtrA1 expression and the differentiation of hepatocellular carcinoma. Loss of HtrA1 was more frequently found in tumors in Edmondson grade especially in those with venous invasion, compared to tumors in Edmondson grade I-II. Most importantly, patients with higher HtrA1 expression had a better survival rate. CONCLUSION: All these data suggest an important role of HtrA1 in hepatocellular carcinoma development and progression, which may be a new target for its treatment.

长链非编码RNA核富集转录本1对瘢痕成纤维细胞增殖、凋亡和迁移的影响

背景:已有研究阐明核富集转录本1(nuclear enriched abundant transcript 1,NEAT1)下调抑制了瘢痕成纤维细胞的进展,但具体机制尚不完全清楚。目的:探讨长链非编码RNA NEAT1调节miR-136-5p/泛素特异性蛋白酶4(ubiquitin specific protease 4,USP4)轴对瘢痕成纤维细胞生物学行为的影响。方法:将瘢痕成纤维细胞分为5组:si-NC组、空白对照组、si-NEAT1组、si-NEAT1+miR-136-5p inhibitor组、si-NEAT1+inhibitor-NC组,qRT-PCR检测NEAT1、miR-136-5p表达;CCK-8法及EDU染色检测细胞增殖能力;流式细胞术检测细胞凋亡情况;划痕愈合实验检测细胞迁移情况;Western blot检测USP4、p27、Bax、基质金属蛋白酶9、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白α1链蛋白表达;双荧光素酶实验检测NEAT1与miR-136-5p、miR-136-5p与USP4的关系。结果与结论:①与si-NC组比较,si-NEAT1组NEAT1表达、A450值、EDU阳性细胞百分比、划痕愈合率以及USP4、基质金属蛋白酶9、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白α1链蛋白表达降低(P<0.05),miR-136-5p表达、细胞凋亡率及p27、Bax蛋白表达升高(P<0.05);②miR-136-5p inhibitor逆转了沉默NEAT1对瘢痕成纤维细胞生物学行为的影响;③miR-136-5p与NEAT1、miR-136-5p与USP4存在靶向调控关系。结果表明,沉默NEAT1可能通过调控miR-136-5p/USP4轴抑制瘢痕成纤维细胞的增殖和迁移,诱导其凋亡。

DNA Methylation Profiles of Protease Nexin 1 (SERPINE2) Gene in Human Cell Lines

Objective： To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1（PN-1） encoded by the SERPINE2 gene in different cell types. Methods： Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. Results： A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2’-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. Conclusion： We have carefully mapped the CpG methylation pattern in two CpG islands in the 5’ part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.

Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury （DAI） at 33℃. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

Synthesis of N1-Substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyra-zolethiocarboxamide as Novel Small Molecule Inhibitors of Cysteine Protease of T.cruzi

A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine protease of T.cruzi..

A new approach for HIV-1 protease cleavage site prediction combined with feature selection

Acquired immunodeficiency syndrome (AIDS) is a fatal disease which highly threatens the health of human being. Human immunodeficiency virus (HIV) is the pathogeny for this disease. Investigating HIV-1 protease cleavage sites can help researchers find or develop protease inhibitors which can restrain the replication of HIV-1, thus resisting AIDS. Feature selection is a new approach for solving the HIV-1 protease cleavage site prediction task and it’s a key point in our research. Comparing with the previous work, there are several advantages in our work. First, a filter method is used to eliminate the redundant features. Second, besides traditional orthogonal encoding (OE), two kinds of newly proposed features extracted by conducting principal component analysis (PCA) and non-linear Fisher transformation (NLF) on AAindex database are used. The two new features are proven to perform better than OE. Third, the data set used here is largely expanded to 1922 samples. Also to improve prediction performance, we conduct parameter optimization for SVM, thus the classifier can obtain better prediction capability. We also fuse the three kinds of features to make sure comprehensive feature representation and improve prediction performance. To effectively evaluate the prediction performance of our method, five parameters, which are much more than previous work, are used to conduct complete comparison. The experimental results of our method show that our method gain better performance than the state of art method. This means that the feature selection combined with feature fusion and classifier parameter optimization can effectively improve HIV-1 cleavage site prediction. Moreover, our work can provide useful help for HIV-1 protease inhibitor developing in the future.

Exploring QSARs for Inhibitory Activity of Cyclic Urea and Nonpeptide-Cyclic Cyanoguanidine Derivatives HIV-1 Protease Inhibitors by Artificial Neural Network

Quantitative structure–activity relationship study using artificial neural network (ANN) methodology were conducted to predict the inhibition constants of 127 symmetrical and unsymmetrical cyclic urea and cyclic cyanoguanidine derivatives containing different substituent groups such as: benzyl, isopropyl, 4-hydroxybenzyl, ketone, oxime, pyrazole, imidazole, triazole and having anti-HIV-1 protease activities. The results obtained by artificial neural network give advanced regression models with good prediction ability. The two optimal artificial neural network models obtained have coefficients of determination of 0.746 and 0.756. The lowest prediction’s root mean square error obtained is 0.607. Artificial neural networks provide improved models for heterogeneous data sets without splitting them into families. Both the external and cross-validation methods are used to validate the performances of the resulting models. Randomization test is employed to check the suitability of the models.

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

A protease-activated receptor 1 antagonist protects against global cerebral ischemia/reperfusion injury after asphyxial cardiac arrest in rabbits

Cerebral ischemia/reperfusion injury is partially mediated by thrombin, which causes brain damage through protease-activated receptor 1（PAR1）. However, the role and mechanisms underlying the effects of PAR1 activation require further elucidation. Therefore, the present study investigated the effects of the PAR1 antagonist SCH79797 in a rabbit model of global cerebral ischemia induced by cardiac arrest. SCH79797 was intravenously administered 10 minutes after the model was established. Forty-eight hours later, compared with those administered saline, rabbits receiving SCH79797 showed markedly decreased neuronal damage as assessed by serum neuron specific enolase levels and less neurological dysfunction as determined using cerebral performance category scores. Additionally, in the hippocampus, cell apoptosis, polymorphonuclear cell infiltration, and c-Jun levels were decreased, whereas extracellular signal-regulated kinase phosphorylation levels were increased. All of these changes were inhibited by the intravenous administration of the phosphoinositide 3-kinase/Akt pathway inhibitor LY29004（3 mg/kg） 10 minutes before the SCH79797 intervention. These findings suggest that SCH79797 mitigates brain injury via anti-inflammatory and anti-apoptotic effects, possibly by modulating the extracellular signal-regulated kinase, c-Jun N-terminal kinase/c-Jun and phosphoinositide 3-kinase/Akt pathways.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

人T细胞白血病病毒1型/人嗜T细胞病毒1型(HTLV-1)及其与相关疾病关系的研究进展

人T细胞白血病病毒1型/人嗜T细胞病毒1型(HTLV-1),是一种和人类T淋巴细胞白血病及HTLV-1相关性脊髓病/热带痉挛性瘫痪(HAM/TSP)等疾病密切相关的逆转录病毒。以往对于该病毒的认识仅仅局限于其导致这两种常见疾病,近来发现越来越多的自身免疫性疾病与该病毒的感染有着密切的联系。同时,该病毒通常经过漫长的潜伏期后发病,一旦发病治疗往往收效甚微,明确病毒如何侵入机体、其致病机制、如何和机体的免疫系统相互作用在细胞内长期潜伏,以便采取有效的早期干预措施将是后续研究的重点方向。我们总结了该病毒导致的相关免疫性疾病、该病毒和免疫系统相互作用的致病机制及治疗方式。

云南HTLV-1成人T细胞白血病1例

成人Ｔ细胞白血病（ＡＴＬ）是流行于日本西南部、加勒比海地区及非洲中部的一种特殊类型的白血病，我国台湾、东南沿海、北京有报道，既往云南尚未报道，我们近来发现１例，现报告如下。１病例介绍患者，男性，３２岁，汉族，云南籍。因头昏、低热、腹胀一月，高热２ｄ于...

HTLV-1蛋白酶与印地那韦识别的分子动力学模拟

对HTLV-1蛋白酶和抑制剂印地那韦的复合物进行了11 ns的分子动力学模拟,从分子整体、氢键、结合自由能及运动性等方面进行了分析。结果表明,动力学模拟使得体系更为松弛,但大部分氢键仍得到保持;疏水相互作用有利于HTLV-1蛋白酶和印地那韦的结合,关键残基主要分布在R10、L30-V39、V56-F67和W98-I1004个区域;结合自由能预测值与试验数据吻合较好。印地那韦结合仅能部分抑制HTLV-1蛋白酶的Flap、top-site以及两个exo-site位点柔性,解释了印地那韦的HIV-1 PR抑制活性为何略大于HTLV-1 PR。该研究为基于受体结构的抗HTLV-1药物研发打下了一定的结构和理论基础。

HTLV-1 Tax蛋白与T细胞的细胞周期调变

人1型T细胞白血病病毒(HTLV-1)能引发成人急性T细胞白血病(ATL)以及一种慢性渐进性的中枢神经系统疾病--热性痉挛性下身截瘫/白血病病毒相关脊髓病(TSP/HAM).成人T细胞白血病(ATL)表现为成熟T淋巴细胞恶性增生.由于HTLV-1 Tax蛋白与T细胞增殖调控有重要关系,本文将主要综述HTLV-1 Tax蛋白如何参与调变T细胞细胞周期从而探讨Tax在T淋巴细胞转化中的作用.

一类具有两个离散时滞的HTLV-1病毒模型动力学分析

目的:研究一类具有细胞内时滞和CTL免疫时滞的HTLV-1病毒动力学行为.方法:定义依赖于时滞的基本再生数R0,建立一个李雅普诺夫函数研究未感染平衡点稳定性,利用特征方程根是否穿越虚轴判断感染平衡点的稳定性.结论:当R0<1时,未感染平衡点是全局稳定的;当R0> 1时,非感染平衡点存在,存在以下两类情形:(a)仅考虑细胞内时滞,非感染平衡点在一定条件下是局部渐近稳定的;(b)仅考虑免疫时滞,系统会产生Hopf分岔.结论:数值模拟验证模型结论有效,可为HTLV-1药物研发提供依据.

MicroRNA-137-5p靶向USP30改善阿尔茨海默病

目的 探索miR-137-5p对阿尔茨海默病(AD)的保护机制。方法 首先用qRT-PCR评估AD患者和健康对照组人血清中miR-137和USP30的表达。用D-半乳糖和氯化铝建立AD小鼠模型,用水迷宫试验检测小鼠的行为,确认AD小鼠模型的成功。用Aβ1-42寡聚体诱导的SH-SY5Y细胞建立AD细胞模型,通过实时定量聚合酶链反应检测AD模型中miR-137-5p和USP30的表达。双重荧光素酶试验用于验证miR-137-5p和USP30之间的靶向结合关系。结果 miR-137-5p的表达在AD患者中与健康对照组相比有所下降(P<0.05),而USP30则明显增加(P<0.05)。miR-137-5p能改善AD细胞模型中的细胞凋亡,USP30的过表达部分废除了miR-137-5p对Aβ1-42-处理的SH-SY5Y细胞的影响,miR-137-5p通过靶向USP30改善AD小鼠的认知能力和Aβ的沉积。结论 miR-137-5p可以通过下调USP30来改善AD症状,miR-137-5p可能能成为治疗AD的一个靶点。