WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1-CUL4-ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5-DDB1-CUL4-ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.     

Chk1及其抑制剂调控肿瘤作用机制研究

细胞周期检查点激酶1（Chk1）是由抑癌基因Chk1编码的丝氨酸/苏氨酸（Ser/Thr激酶）,广泛存在于哺乳动物细胞内。近年来,多项国内外研究表明Chk1及其抑制剂能调控肿瘤的形成与发展。基于此,本文以Chk1及其抑制剂的概况和应用为基础,总结其多向调控肿瘤细胞的作用机制,旨在为Chk1及其抑制剂的临床应用和开发、相关药物的研发提供参考。     

泛素连接酶LNX1促进外源PBK的泛素化和降解

 目的 研究LNX1对其相互作用蛋白PBK的泛素化和降解。方法 克隆、原核表达、纯化了一系列重组人LNX1截断体蛋白和LNX1全长蛋白;在体外泛素化体系中研究其对PBK的泛素化,哺乳动物细胞内研究其对外源PBK的泛素化和降解。结果 在体外泛素化体系中LNX1泛素化PBK,并研究了不同LNX1截断体对PBK泛素化的影响;发现在哺乳动物细胞内外源LNX1促进外源PBK的泛素化,进而导致其通过蛋白酶体降解。结论 研究发现了LNX1对外源PBK的泛素化和降解,为研究LNX1的生理功能提供了重要线索。     

Mutations at multiple CDK phosphorylation consensus sites on Cdt2 increase the affinity of CRL4Cdt2 for PCNA and its ubiquitination activity in S phase

CRL4^Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4^Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C‐terminal half region of Cdt2 (Cdt2‐18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2‐18A was colocalized with PCNA foci during S phase in comparison with Cdt2‐WT. Poly‐ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2‐18A. Other CRL4^Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2‐18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK‐mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.     

The multifaceted role of the E3 ubiquitin ligase HOIL-1: beyond linear ubiquitination

Ubiquitination controls and fine-tunes many signaling processes driving immunity, inflammation, and cancer. The E3 ubiquitin ligase HOIL-1 (heme-oxidized IRP2 ubiquitin ligase-1) is increasingly implicated in different signaling pathways and plays a vital role in immune regulation. HOIL-1 co operates with the E3 ubiquitin ligase HOIP (HOIL-1 interacting protein) to modify specific nuclear factor-κB (NF-κB) signaling proteins with linear M1-linked polyubiquitin chains. In addition, through its ability to also add K48-linked polyubiquitin chains to specific substrates, HOIL-1 has been linked with antiviral signaling, iron and xenobiotic metabolism, cell death, and cancer. HOIL-1 deficiency in humans leads to myopathy, amylopectinosis, auto-inflammation, and immunodeficiency associated with an increased frequency of bacterial infections. HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium, pathogen-specific immunodeficiency, but minimal signs of hyper-inflammation. This review summarizes current knowledge on the mechanism of action of HOIL-1 and highlights recent advances regarding its role in health and disease.     

前列腺癌p21CIP1/WAF1、Rb及PCNA的表达及意义

目的研究p21^CIP1/WAF1、Rb及PCNA在人前列腺癌标本中的表达及三者相关性,阐述它们与前列腺癌病理分级及临床分期的关系。方法收集36例确诊前列腺癌石蜡包埋存档标本作为研究对象,采用免疫组化SABC法对其p21^CIP1/WAF1、Rb及PCNA进行检测,并应用图象分析仪判定,统计学处理,对前列腺癌的病理分级及临床分期进行了对比分析及相关性研究。结果p21^CIP1/WAF1、Rb及PCNA的免疫组化阳性染色为棕黄色和／或棕褐色,定位于细胞浆或细胞核。所得数据均经统计学处理。在不同病理分级、临床分期之间差异均有显著性,同时还发现PCNA与p21^CIP1/WAF1及Rb之间存在显著负相关性。但未发现p21^CIP1/WAF1、与Rb有显著相关性。结论p21^CIP1/WAF1、Rb表达与前列腺癌组织的病理分级、临床分期呈负相关性,在发病机制中可能涉及到p21^CIP1/WAF1、Rb和PCNA凋节通路的异常。同时,作为一种检测方法,可用于判定前列腺癌恶性程度及进程的有价值的指标,对诊治康复也可提供有意义的帮助。     

CyclinD1和PCNA在子宫内膜癌中表达及临床意义

目的探讨CycliD1和PCNA在子宫内膜癌中的表达及临床意义。方法应用免疫组化法检测50例子宫内膜癌,22例子宫内膜不典型增生和8例正常子宫内膜中CyclinD1,PCNA的表达。结果 CyclinD1和PCNA在子宫内膜癌中的阳性表达率分别为54%(27/50)和68%(34/50),明显高于正常子宫内膜组(P0.05)。子宫内膜癌中,CyclinD1的表达与FIGO分期和淋巴结转移密切相关(P0.05),PCNA的表达与病理分级和淋巴结转移密切相关(P0.05)。相关性分析显示,CyclinD1和PCNA在子宫内膜癌中的表达呈正相关(r=0.742,P0.05)。二者协同异常表达者生物学行为较好。结论 CyclinD1和PCNA可能在子宫内膜癌发生发展中起重要作用,可作为评估预后的指标。     

The oncogene ABL1 regulates the inflammatory response of innate immunity via mediating TRAF6 ubiquitination

The oncogene ABL1 plays an important role in various cancers, while its roles remain unclear in pneumonia. This study aims to investigate the roles of ABL1 in pneumonia and the underlying mechanisms. RNA sequencing was used to determine the expressions of multiple kinases in the PBMCs. A series of overexpression and knockout cell lines were constructed. Besides, an intranasal lung infection mouse model was pre-treated with asciminb. ELISAs and qPCR were used to determine the levels of target genes. In addition, STRING Interaction Network and Immunoblotting assays were used to determine the interaction between target proteins. An elevation in ABL1 was observed in the infant with Ecoli pneumonia. ABL1 was positively correlated to the levels of inflammatory cytokines and the activation of the NF-kB pathways. In vivo data demonstrated that the inhibition of ABL1 suppressed the inflammatory cytokines, reduced the lung bacterial burden, and ameliorated the lung injury score. ABL1 inhibited the phosphorylation of IκBα and p38 and regulated the ubiquitination of TRAF6. ABL1 regulates the inflammatory response in pneumonia in part by the regulation of MAPK and NF-κB pathways and TRAF6 ubiquitination.     

子宫内膜腺癌组织中cyclinD1、PCNA和Ki-67的表达

目的　探讨cyclinD1、PCNA和Ki 6 7在子宫内膜腺癌组织中的表达及其意义。 方法　采用免疫组化S P法检测正常增生期子宫内膜 10例、单纯性增生 30例、复杂性增生 30例、非典型增生 30例和子宫内膜腺癌 4 7例组织中cyclinD1、PCNA和Ki 6 7的表达。结果　cyclinD1在增生期子宫内膜组织中未见阳性表达 ,在单纯性增生、复杂性增生、非典型增生和子宫内膜腺癌组织中的阳性表达率分别为 13 3%、16 7%、30 0 %和 5 7 8%。非典型增生和子宫内膜腺癌组织阳性表达率高于增生期宫内膜、单纯性和复杂性增生 (P <0 0 5 ) ,cyclinD1蛋白表达与子宫内膜腺癌临床分期、肌层浸润和淋巴结转移呈正相关 (P<0 0 5 ) ,而与肿瘤的分化程度无关 (P >0 0 5 )。PCNA和Ki 6 7在非典型增生组标记指数 (LI)高于增生期宫内膜、单纯性和复杂性增生组 ,子宫内膜腺癌组高于非典型增生组 (P <0 0 5 ) ,PCNA和Ki 6 7LI与肿瘤的分化程度、临床分期、肌层浸润和淋巴结转移呈正相关 (P <0 0 5 )。PCNA和Ki 6 7在cyclinD1阳性表达组的LI高于阴性组 (P <0 0 5 )。结论　cyclinD1蛋白过度表达可能在子宫内膜癌的发生、发展中起重要作用 ,其作用途径可能是通过促进细胞增殖而实现的。     

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks.     

Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep)

Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery. Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication. It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR). By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication. We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA). Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep. We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.     

MIB-1 and PCNA immunostaining as a diagnostic adjunct to cervical Pap smear

The present study was done to determine the role of MIB-1 (Molecular Immunology Borstel) and proliferating cell nuclear antigen (PCNA) proliferative index as a diagnostic adjunct to cervical Papanicolaou (Pap) smear for the identification of ascending grades of cervical intraepithelial neoplasia (CIN) developing into cancer in the human uterine cervix. A total of 49 adequate Pap smears with consensus diagnosis were destained for immunocytochemical staining (MIB-1 and PC10). Staining was done by streptavidin-biotin method after antigen retrieval. MIB-1 and PC10 labeling index (LI) were calculated in each case and divided into three groups, i.e., <10%, 10-20%, and >20%, respectively. Statistical analysis was done by using the SPSS 10.0 package. The comparisons were made using analysis of variance (ANOVA) and independent sample t-test. Bivariate and Pearson's correlation coefficient were used to obtain correlations between different groups. Out of 49 cases, 40 cases (81.6%) showed positive immunostaining with MIB-1 and PCNA. Proliferative LI of MIB-1 and PCNA increased with the ascending grades of CIN lesions to carcinoma. The highest proliferative index (mean +/- SD) for PCNA and MIB-1 were observed for the carcinoma group (PCNA LI, 39.200 +/- 1.6865; MIB-1LI, 35.300 +/- 1.8886). A significant positive correlation between ascending grades of squamous intraepithelial lesion (SIL) and labeling indices of markers (r = 0.87 for MIB-1 and r = 0.88 for PCNA) suggests that MIB-1/PCNA proliferative markers can be used as an adjunct to cytomorphological interpretation of conventional cervical Pap smear.     

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

The ATR–CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA–PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.     

Cyclin D1和p16、p27在脑胶质瘤中的表达及其与PCNA的相关性

目的 :检测CyclinD1 和p16、p2 7以及PCNA在脑胶质瘤中的表达状况 ,以探讨不同病理类型脑胶质瘤中各自的表达及其相关性。方法 :应用免疫组化S P法 ,检测 12例正常脑组织、5 8例脑胶质瘤组织中CyclinD1 、p16、p2 7及PCNA表达及其特征。结果 :CyclinD1 在由低度恶性胶质瘤向高度恶性胶质瘤转化中阳性表达逐渐增强 (χ2 检验 ,P <0 0 0 5 ,χ2 =5 1 6 7) ;而p16、p2 7阳性表达却是随着胶质瘤恶性程度的升高而降低 (χ2 检验 ,P值均 <0 0 0 5 ,χ2 分别为 15 4 1和 12 81)。CyclinD1 与PCNA呈正相关 ,rs =0 74 5 ;p16和p2 7与PCNA呈负相关 ,rs分别为 - 0 5 6 6和 - 0 6 12。结论 :脑胶质瘤中CyclinD1的表达程度对胶质瘤的细胞增殖活性起促进作用 ,而p16、p2 7的表达则起抑制作用。     

Characterisation of the interaction between WRN,the helicase/exonuclease defective in progeroid Werner's syndrome,and an essential replication factor,PCNA

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.     

Distributional changes of BrdU, PCNA, E2F1 and PAL31 molecules in developing murine palatal rugae

The distribution of cells incorporating bromodeoxyuridine (BrdU) and the expression of molecules involved in the control of cell proliferation (proliferating cell nuclear antigen [PCNA], a cellular factor in F9 teratocarcinoma cells that recognizes an adenovirus E1A inducible promoter 1 [E2F1] and proliferation-related acidic nuclear protein 31 [PAL31]) during morphogenesis of the murine palatine rugae (PR) was examined histochemically. Pattern formation of the PR rudiment was initiated with cell cycle related molecules in the epithelium of the primary palate. Cells which had incorporated BrdU were detected at the outer areas of the presumptive epithelial placode (EP) and the EP at 11.5–13.5 days post coitum (dpc) and the outer areas of the PR protrusion after 14.5 dpc. The number of PCNA-positive cells at the central area of the PR protrusion decreased after 16.5 dpc. E2F-positive cells were detected at the outer areas of the PR protrusion at 15.5 and 16.5 dpc. The number of PAL31-positive cells at the presumptive EP area and the already-formed EP area was decreased at 11.5–13.5 dpc. In two dimensional histological reconstructions, PAL31 expression approximately corresponded to the distribution of BrdU-positive cells at 11.5 and 13.5 dpc. EP placode formation might be regulated by spatiotemporal cell proliferation control involving the expression of the PAL31 molecule. Following EP formation, PR development and growth control involved the expression of E2F1 and PCNA molecules.     

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.