拟素化酶和去拟素化酶在肺癌发生发展中的作用

蛋白质拟素化是一种类似于泛素化的翻译后修饰，由NEDD8活化酶E1 (NAE)、NEDD8耦联酶E2 (UBE2M或UBE2F)和NEDD8连接酶E3三种酶催化组成的级联反应。Cullin家族蛋白是拟素化修饰的生理性底物，Cullin的拟素化修饰激活Cullin-RING连接酶（CRLs）,CRLs是最大一类E3泛素连接酶家族，介导了其中约20%蛋白质的泛素化降解来调节许多生物过程，包括细胞周期调控、DNA损伤修复、细胞生长、代谢、存活、自噬、迁移和免疫逃逸等。去拟素化过程则是通过特异性的去拟素化酶将拟素分子NEDD8从底物蛋白上水解并移除，释放至细胞中以维持拟素化的动态平衡。NEDD8和拟素化修饰的催化酶在多种癌症中高表达或活性上调，导致CRLs的过度激活，催化许多抑癌蛋白质的降解，从而促进肺癌细胞的增殖与存活以及肺肿瘤的发生发展。蛋白质拟素化修饰已被证实是有希望的癌症靶点。同样地，多种去拟素化酶在肺癌中高表达，其改变也与多种恶性肿瘤的发生发展密切相关，亦是潜在的肿瘤治疗重要靶点。本综述主要聚焦于拟素化及去拟素化通路在肺癌细胞中表达水平的改变，如何调节肺癌细胞的生长、存活和肺癌微环境...     

Biochemical and cellular effects of inhibiting Nedd8 conjugation

The conjugation of proteins with the ubiquitin-like protein Nedd8 is an essential cellular process and an important anti-cancer therapeutic target. The major known role of Nedd8 is the attachment to and activation of Cullin RING E3 ubiquitin ligases (CRL). The attachment of Nedd8 to its substrates occurs via a process analogous to ubiquitin transfer, involving a Nedd8 E1 activating enzyme and a Nedd8 E2 conjugating enzyme, Ubc12, which transfers Nedd8 onto lysine residues of target proteins. In this study, we utilize dominant-negative Ubc12 (dnUbc12) and the Nedd8 E1 inhibitor MLN4924 to inhibit cellular neddylation. We demonstrate that dnUbc12 functions by depleting cellular Nedd8 concentrations. Inhibition of cellular neddylation leads to rapid accumulation of CRL substrates and an enlarged and flattened morphology in HEK293 cells. Inhibiting Nedd8 conjugation also causes abnormalities in the actin cytoskeleton. This is likely at least partially mediated via accumulation of the small GTPase RhoA, a recently identified CRL substrate. We indeed found that siRNA mediated knockdown of RhoA can reverse the morphological changes observed upon inhibition of cellular neddylation. In conclusion, the Nedd8 pathway plays an important role in regulating the actin cytoskeleton and cellular morphology. Dysfunction of the actin cytoskeleton may contribute to the anti-cancer effect of Nedd8 inhibition.     

MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes

The protein MDC1/NFBD1 contains a forkhead-associated (FHA) domain and two BRCA1 carboxyl-terminal (BRCT) domains. It interacts with several proteins involved in DNA damage repair and checkpoint signalling, and is phosphorylated in response to DNA damage and during mitosis. Upon treatment of cultured human cells with DNA damaging agents, MDC1/NFBD1 translocates to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci. Down-regulation of MDC1/NFBD1 expression levels by small interfering RNA (siRNA) renders cells hyper-sensitive to DNA damaging agents and leads to defects in cell cycle checkpoint activation and apoptosis. Thus, MDC1/NFBD1 appears to be a key regulator of the DNA damage response in mammalian cells.     

Neddylation-induced conformational control regulates cullin RING ligase activity in vivo

Cullin RING ligases (CRLs) constitute the largest family of ubiquitin ligases with diverse cellular functions. Conjugation of the ubiquitin-like molecule Nedd8 to a conserved lysine residue on the cullin scaffold is essential for the activity of CRLs. Using structural studies and in vitro assays, it has been demonstrated that neddylation stimulates CRL activity through conformational rearrangement of the cullin C-terminal winged-helix B domain and Rbx1 RING subdomain from a closed architecture to an open and dynamic structure, thus promoting ubiquitin transfer onto the substrate. Here, we tested whether the proposed mechanism operates in vivo in intact cells and applies to other CRL family members. To inhibit cellular neddylation, we used a cell line with tetracycline-inducible expression of a dominant-negative form of the Nedd8 E2 enzyme or treatment of cells with the Nedd8 E1 inhibitor MLN4924. Using these cellular systems, we show that different mutants of Cul2 and Cul3 and of Rbx1 that confer increased Rbx1 flexibility mimic neddylation and rescue CRL activity in intact cells. Our findings indicate that in vivo neddylation functions by inducing conformational changes in the C-terminal domain of Cul2 and Cul3 that free the RING domain of Rbx1 and bridge the gap for ubiquitin transfer onto the substrate.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

Analysis of DNA repair and recombination responses in mouse cells depleted for Brca2 by SiRNA

The tumor suppressor BRCA2 is considered to play an important role in the maintenance of genome integrity through the repair of DNA lesions by homologous recombination. A mechanistic understanding of BRCA2 has been complicated by the embryonic lethality of mice bearing allelic knockouts of Brca2, and by variation in the DNA damage response in cells bearing BRCA2 deficiencies. It would be advantageous to develop approaches that avoid the cell lethality associated with complete inactivation of the gene, or the use of established tumor cell lines in which other genes in addition to BRCA2 may be mutant. In this study, SiRNA was used in stable transformation assays to knockdown Brca2 in mouse hybridoma cells by at least 75%. The Brca2-depleted cells were analyzed with respect to cell growth, sensitivity to DNA damaging agents (mitomycin C, methylmethane sulfonate, or ionizing radiation), intrachromosomal homologous recombination and gene targeting. Although the effect of Brca2-depletion on cell growth and sensitivity to DNA damaging agents was modest, the Brca2-depleted cells did show a significant shift in homologous recombination from gene conversion to single-strand annealing and a significant decrease in the efficiency of gene targeting. Both of these phenotypes are consistent with the proposed role of Brca2 in DNA repair and recombination.     

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.     

USP7/HAUSP stimulates repair of oxidative DNA lesions

USP7 is involved in the cellular stress response by regulating Mdm2 and p53 protein levels following severe DNA damage. In addition to this, USP7 may also play a role in chromatin remodelling by direct deubiquitylation of histones, as well as indirectly by regulating the cellular levels of E3 ubiquitin ligases involved in histone ubiquitylation. Here, we provide new evidence that USP7 modulated chromatin remodelling is important for base excision repair of oxidative lesions. We show that transient USP7 siRNA knockdown did not change the levels or activity of base excision repair enzymes, but significantly reduced chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.     

Cullin neddylation and substrate‐adaptors counteract SCF inhibition by the CAND1‐like protein Lag2 in Saccharomyces cerevisiae

Cullin‐based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin‐like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non‐neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C‐terminal β‐hairpin structure that inserts into the Skp1‐binding pocket on the cullin, and an N‐terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N‐terminal‐binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate‐specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re‐association of Lag2/Cand1.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.     

Neddylation dysfunction in Alzheimer's disease

Ubiquitin‐dependent proteolysis is a major mechanism that downregulates misfolded proteins or those that have finished a programmed task. In the last two decades, neddylation has emerged as a major regulatory pathway for ubiquitination. Central to the neddylation pathway is the amyloid precursor protein (APP)‐binding protein APP‐BP1, which together with Uba3, plays an analogous role to the ubiquitin‐activating enzyme E1 in nedd8 activation. Activated nedd8 covalently modifies and activates a major class of ubiquitin ligases called Cullin‐RING ligases (CRLs). New evidence suggests that neddylation also modifies Type‐1 transmembrane receptors such as APP. Here we review the functions of neddylation and summarize evidence suggesting that dysfunction of neddylation is involved in Alzheimer's disease.     

SOS regulatory elements are essential for UPEC pathogenesis

Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells. LexA and RecA coordinate various operons for repair of DNA damage due to exogenous and endogenous agents and are known regulators of sulA. UPEC strains defective in regulation of the SOS response mediated by RecA and LexA display attenuated virulence in immunocompetent mice within the first 6 h post infection. RecA and LexA regulation of the SOS regulon is dispensable in immunocompromised mice. These data suggest that epithelial cells produce sufficient levels of DNA damaging agents, such that the bacterial DNA damage repair response is essential, as a means to control invading bacteria. Since many pathogens interact with the epithelium before exposure to professional phagocytes, it is likely that adaptation to oxidative radicals during intracellular growth provides additional protection from killing by innate immune phagocytes.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

Viral transport of DNA damage that mimics a stalled replication fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.