An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100kb), rare copy number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1,564 cases and 1,748 controls all from Ireland, and further extended the analysis to include an additional 5,196 UK controls. We found association with duplications at chr20p12.2 (P=0.007) and evidence of replication in large independent European schizophrenia (P=0.052) and UK bipolar disorder case-control cohorts (P=0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11,707 cases and 10 carriers in 21,204 controls (meta-analysis CMH P value=2x10(-4) (odds ratio (OR)=11.3, 95% CI=3.7, ∞)). Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68Mb with similar breakpoints across samples. By haplotype analysis and sequencing we identified a tandem ∼149kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P=2.5x10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

A Burkholderia cenocepacia gene encoding a non-functional tyrosine phosphatase is required for the delayed maturation of the bacteria-containing vacuoles in macrophages

Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles (BcCVs) that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low-molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the BCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since BCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in B. multivorans confers to this bacterium a similar phagosomal maturation delay as found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.

S100A2 is a BRCA1/p63 coregulated tumour suppressor gene with roles in the regulation of mutant p53 stability

Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.