Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

The Mitotic Exit Network Mob1p-Dbf2p Kinase Complex Localizes to the Nucleus and Regulates Passenger Protein Localization

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates CDK inactivation, cytokinesis and G1 gene transcription. The MEN Cdc14p phosphatase is sequestered in the nucleolus and transiently released in early anaphase and telophase. Cdc14p mediates mitotic exit by dephosphorylating Cdk1p substrates and promoting Cdk1p inactivation. Cdc14p also regulates the localization of chromosomal passenger proteins, which redistribute from kinetochores to the mitotic spindle during anaphase. Here we present evidence that the MEN protein kinase complex Mob1p-Dbf2p localizes to mitotic nuclei and partially colocalizes with Cdc14p and kinetochore proteins. Chromatin immunoprecipitation (ChIP) experiments reveal that Mob1p, Dbf2p, and Cdc14p associate with centromere DNA and require the centromere binding protein Ndc10p for this association. We establish that Mob1p is essential for maintaining the localization of Aurora, INCENP, and Survivin chromosomal passenger proteins on anaphase spindles, whereas Cdc14p and the Mob1p-Dbf2p-activating kinase Cdc15p are required for establishing passenger protein localization on the spindle. Moreover, Mob1p, but not Cdc15p, is required for dissociating Aurora from the kinetochore region. These findings reveal kinetochores as sites for MEN signaling and implicate MEN in coordinating chromosome segregation and/or spindle integrity with mitotic exit and cytokinesis via regulation of chromosome passenger proteins

SspH2 stability and activity are enhanced by HsSGT1A <i>in vitro</i>.

<p><b>A,</b> Time course of <i>in vitro</i> ubiquitination assay conducted with purified recombinant HA-Ubiquitin, UBE1 (E1), UbcH5b (E2), His-HsSGT1A and GST-HA-SspH2 as indicated. Reactions were quenched at the indicated time and immunoblotted with the indicated antibodies to detect SspH2 activity, SspH2 stability and HsSGT1A ubiquitination. <b>B, </b><i>In vitro</i> ubiquitination assay reaction components were pre-incubated for the indicated times, initiated with HA-ubiquitin and quenched after 2 hours. Samples were immunoblotted as outlined above. Molecular weights are indicated on the left.</p

<i>S.</i> Typhimurium SspH2 does not alter the essential cell cycle function of SGT1.

<p><b>A,</b> Dilution series of wild type yeast expressing empty vector (EV) or SspH2 with HA-epitope tag grown at 25°C. The expression of HA-tagged SspH2 was assessed by immunoblot in total lysate. <b>B,</b> Dilution series of yeast <i>sgt1-3</i> complemented with ScSgt1p (Sc), HsSGT1A (HsA), HsSGT1B (HsB) or empty vector (EV) and expressing SspH2-HA (+) or empty vector (−), were grown at the permissive (25°C) and restrictive (32°C) temperatures. <b>C,</b> The expression of HA-tagged SspH2 in cultures from (<b>B</b>) was assessed by immunoblot in total cell lysate. The membrane was stained with Ponceau S (Pon. S) to indicate protein loading. <b>D,</b> HeLa cells transfected with plasmid control (EV) or constitutive expression vectors for SspH1 or SspH2 were quantified for cytotoxicity. Data are presented as the mean ± standard error of the mean for three independent determinations. <i>S.</i> Typhimurium effectors were tagged with an HA epitope and shown in the inset is the expression of transfected constructs as assessed by α-HA IB. Calnexin is a loading control.</p

<i>S.</i> Typhimurium SspH2 enhances SGT1-dependent NLR immune response in mammalian cells.

<p><b>A</b>, Establishment of a Nod1-mediated IL-8 secretion assay in HeLa cells. Secreted IL-8 was quantified in the presence and absence of transfected Nod1 and Nod1 agonist. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003518#s4" target="_blank">Materials and methods</a> for more detail. <b>B</b>, Effect of <i>S.</i> Typhimurium effector constructs on Nod1-mediated IL-8 secretion. HeLa cells were co-transfected with Nod1 and effector constructs and treated with Nod1 agonist. Data are presented as the mean ± standard error of the mean for three (<b>A</b>) and twelve (<b>B</b>) independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.005 between the indicated sample and the empty vector control (EV) sample. # denotes <i>p</i><0.005 between the indicated samples. <b>C</b>, The expression of epitope tagged <i>S.</i> Typhimurium effectors (HA) and exogenous Nod1 (FLAG) was assessed by immunoblot in total lysate. Calnexin is a loading control. <b>D</b>, Effect of <i>S.</i> Typhimurium infection on IL-8 secretion. HeLa cells were infected with the wild type strain, <i>sspH2</i> deletion mutant (Δ<i>sspH2</i>), and <i>sspH2</i> deletion mutant complemented with empty vector (Δ<i>sspH2</i>+pWSK) or <i>sspH2</i> (Δ<i>sspH2</i>+p<i>sspH</i>2-HA). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003518#s4" target="_blank">Materials and methods</a> for more detail. Data are presented as the mean ± standard error of the mean for six independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.005 between the indicated sample and all other samples. ns, denotes no significant difference between the indicated samples.</p

<i>S.</i> Typhimurium SspH2 enhances SGT1-dependent NLR immune response <i>in planta</i>.

<p><b>A</b>, An NbSGT1-silenced (TRV:SGT) Rx-transgenic <i>N. benthamiana</i> (Nb Rx) leaf was agro-infiltrated with PVX-GFP and also 5xMyc-AtSGT1A (AtA), GUS, SspH2-3xFLAG (H2), SspH1-3xFLAG (H1) and SspH2C580A-3xFLAG (C580A) as indicated. As controls the same infiltrations were performed in a control-silenced (TRV:00) Nb Rx leaf and non-transgenic (Nb WT) TRV:SGT leaf. Leaves were imaged under UV lighting 7 days post-infiltration. <b>B</b>, Infiltration site lysate from samples in (<b>A</b>) were immunoblotted with α-GFP to detect PVX replication. Membranes were stained with Ponceau S (Pon. S) to indicate protein loading. <b>C</b>, Nb Rx TRV:SGT leaf was agro-infiltrated as indicated in (<b>A</b>) and imaged under brightfield 7 days post-infiltration. Higher magnification panels of the brightfield image are provided for comparison.</p

<i>S.</i> Typhimurium SspH2 interacts with and ubiquitinates Nod1 causing agonist-independent activation of Nod1.

<p><b>A,</b> Reciprocal co-IP analyses of Nod1 and SspH2 or SspH1 transiently expressed in HEK 293T cells. <i>S.</i> Typhimurium effectors and Nod1 were tagged with HA and FLAG epitopes, respectively. IPs and immunoblotting (IB) were performed with the indicated antibodies. <b>B,</b> Co-IP analysis of transiently expressed Nod1 and either SspH2 or SspH2C580A and endogenous SGT1 in HEK 293T cells. SspH2 constructs and Nod1 were tagged with HA and FLAG epitopes, respectively. IPs and IB were performed with the indicated antibodies. <b>C,</b> Effect of <i>S.</i> Typhimurium effector constructs on Nod1-mediated IL-8 secretion. HeLa cells were co-transfected with Nod1 and effector constructs but not treated with Nod1 agonist. Data are presented as the mean ± standard error of the mean for three independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.005 between the SspH2 and empty vector control (EV) samples. <i>S.</i> Typhimurium effectors and Nod1 were tagged with HA and FLAG epitopes, respectively. Shown in the inset is the expression of transfected constructs as assessed by IB with the indicated antibodies. Calnexin is a loading control. <b>D,</b> Effect of proteasome inhibitor on SspH2-mediated enhancement of IL-8 secretion. HeLa cells were co-transfected with Nod1 and SspH2 or SspH2C580A and treated with MG132 or vehicle during Nod1 agonist addition. Data are presented as the mean ± standard error of the mean for eight independent determinations. Data were analyzed using a non-parametric Mann-Whitney test and * denotes <i>p</i><0.0005 between the SspH2 and SspH2C580A control samples. SspH2 constructs and Nod1 were tagged with HA and FLAG epitopes, respectively. Shown in the inset is the expression of transfected constructs as assessed by IB with the indicated antibodies. GAPDH is a loading control. <b>E,</b> Co-IP analyses of Nod1, ubiquitin and SspH2 or SspH2C580A transiently expressed in HEK 293T cells. SspH2 constructs, Nod1 and ubiquitin were tagged with HA, FLAG and Myc epitopes, respectively. IPs and IB were performed with the indicated antibodies. The α-Flag IP samples were co-immunoblotted against α-Flag and α-Myc antibodies and detected in separate channels, confirming that the Nod1 and ubiquitin signals overlaid at the indicated band (*).</p

Synthetic Lethal Genetic Interactions That Decrease Somatic Cell Proliferation in Caenorhabditis elegans Identify the Alternative RFCCTF18 as a Candidate Cancer Drug Target

Somatic mutations causing chromosome instability (CIN) in tumors can be exploited for selective killing of cancer cells by knockdown of second-site genes causing synthetic lethality. We tested and statistically validated synthetic lethal (SL) interactions between mutations in six Saccharomyces cerevisiae CIN genes orthologous to genes mutated in colon tumors and five additional CIN genes. To identify which SL interactions are conserved in higher organisms and represent potential chemotherapeutic targets, we developed an assay system in Caenorhabditis elegans to test genetic interactions causing synthetic proliferation defects in somatic cells. We made use of postembryonic RNA interference and the vulval cell lineage of C. elegans as a readout for somatic cell proliferation defects. We identified SL interactions between members of the cohesin complex and CTF4, RAD27, and components of the alternative RFCCTF18 complex. The genetic interactions tested are highly conserved between S. cerevisiae and C. elegans and suggest that the alternative RFC components DCC1, CTF8, and CTF18 are ideal therapeutic targets because of their mild phenotype when knocked down singly in C. elegans. Furthermore, the C. elegans assay system will contribute to our knowledge of genetic interactions in a multicellular animal and is a powerful approach to identify new cancer therapeutic targets