Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

Role of oculocerebrorenal syndrome of Lowe (OCRL) protein in megakaryocyte maturation, platelet production and functions: a study in patients with Lowe syndrome.

Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.

MTMR14 protects against hepatic ischemia-reperfusion injury through interacting with AKT signaling in vivo and in vitro.

Hepatic ischemia-reperfusion (IR) injury is characterized by severe inflammation and cell death. However, very few effective therapies are presently available for hepatic IR injury treatment. Here, we reported a protective function and the underlying mechanism of myotubularin-related protein 14 (MTMR14) during hepatic IR injury. Hepatocyte-specific MTMR14 knockout (HKO) and transgenic (TG) mice were subjected to hepatic IR operation to explore MTMR14 function in vivo. Primary hepatocytes isolated from MTMR14-HKO and MTMR14-TG mice were subjected to hypoxia/reoxygenation (HR) insult in vitro. We found that MTMR14 expression in liver tissues from individuals with hepatic IR was markedly decreased, and similar results were detected in mice with hepatic IR surgery. MTMR14-TG mice following hepatic IR operation had obviously ameliorated liver pathological changes, along with improved hepatic dysfunction, which was proved by the decreased serum alanine amino transferase (ALT) and aspartate amino transferase (AST) levels. MTMR14-HKO and MTMR14-TG animal models indicated that MTMR14 alleviated cell death and inflammatory response. In addition, MTMR14 inhibited nuclear transcription factor κB (NF-κB) signaling. Of note, promoting MTMR14 expression improved phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT) pathway through a physical interaction with AKT, subsequently reducing cell death and inflammation. Therefore, MTMR14 is a protective factor during hepatic IR injury, and the MTMR14/AKT signaling is involved the pathogenesis hepatic IR injury. Improvement of this axis might be a novel therapeutic strategy for the prevention of this pathological process.

The Effect of TIGAR Knockdown on Apoptotic and Epithelial-Mesenchymal Markers Expression in Doxorubicin-Resistant Non-Small Cell Lung Cancer A549 Cell Lines.

Resistance to chemotherapeutic drugs is a critical problem in cancer therapy, but the underlying mechanism has not been fully elucidated. TP53-induced glycolysis regulatory phosphatase (TIGAR), an important glycolysis and apoptosis regulator, plays a crucial role in cancer cell survival by protecting cells against oxidative stress-induced apoptosis. In the present study, we investigated whether TIGAR is involved in epithelial-mesenchymal transition (EMT) in doxorubicin (DOX)-resistant human non-small cell lung cancer (NSCLC), A549/DOX cells. We found that the expression of TIGAR was significantly higher in A549/DOX cells than in the parent A549 cell lines. siRNA-mediated TIGAR knockdown reduced migration, viability and colony survival of doxorubicin-resistant lung cancer cells. Also, TIGAR knockdown decreased pro-survival protein Bcl-2 and increased pro-apoptotic Bax and cleaved poly (ADP-ribose) polymerase (PARP). Moreover, TIGAR depletion significantly up-regulated both caspase-3 and caspase-9 expression. Furthermore, TIGAR depletion up-regulated the expression of E-cadherin and down-regulated the expression of vimentin. These results indicate that TIGAR knockdown may inhibit EMT in doxorubicin (DOX)-resistant human NSCLC and may represent a therapeutic target for a non-small lung cancer cells chemoresistance.

Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment.

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

Mth1 deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice.

Exposure to asbestos fiber is central to mesothelial carcinogenesis. Recent sequencing studies on human and rodent malignant mesothelioma (MM) revealed frequently mutated genes, including CDKN2A, BAP1 and NF2. Crocidolite directly or indirectly catalyses the generation of hydroxyl radicals, which appears to be the major driving force for mesothelial mutations. DNA base modification is an oxidative DNA damage mechanism, where 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the most abundant modification both physiologically and pathologically. Multiple distinct mechanisms work together to decrease the genomic level of 8-OHdG through the enzymatic activities of Mutyh, Ogg1 and Mth1. Knockout of one or multiple enzymes is not lethal but increases the incidence of tumors. Here, we used single knockout (KO) mice to test whether the deficiency of these three genes affects the incidence and prognosis of asbestos-induced MM. Intraperitoneal injection of 3 mg crocidolite induced MM at a fraction of 14.8% (4/27) in Mth1 KO, 41.4% (12/29) in Mutyh KO and 24.0% (6/25) in Ogg1 KO mice, whereas 31.7% (20/63) induction was observed in C57BL/6 wild-type (Wt) mice. The lifespan of female Mth1 KO mice was longer than that of female Wt mice (p = 0.0468). Whole genome scanning of MM with array-based comparative genomic hybridization revealed rare genomic alterations compared to MM in rats and humans. These results indicate that neither Mutyh deficiency nor Ogg1 deficiency promotes crocidolite-induced MM in mice, but the sanitizing nucleotide pool with Mth1 is advantageous in crocidolite-induced mesothelial carcinogenesis.

Myotubularin-related protein 14 suppresses cardiac hypertrophy by inhibiting Akt.

Cardiac hypertrophy (CH) is an independent risk factor for many cardiovascular diseases, and is one of the primary causes of morbidity and mortality in elderly people. Pathological CH involves excessive protein synthesis, increased cardiomyocyte size, and ultimately the development of heart failure. Myotubularin-related protein 14 (MTMR14) is a member of the myotubularin (MTM)-related protein family, which is involved in apoptosis, aging, inflammation, and autophagy. However, its exact function in CH is still unclear. Herein, we investigated the roles of MTMR14 in CH. We show that MTMR14 expression was increased in hypertrophic mouse hearts. Mice deficient in heart MTMR14 exhibited an aggravated aortic-banding (AB)-induced CH phenotype. In contrast, MTMR14 overexpression prevented pressure overload-induced hypertrophy. At the molecular level, prevention of CH in the absence of MTMR14 involved elevations in Akt pathway components, which are key elements that regulate apoptosis and cell proliferation. These results demonstrate that MTMR14 is a new molecular target for the treatment of CH.

Increased MTH1-specific 8-oxodGTPase activity is a hallmark of cancer in colon, lung and pancreatic tissue.

Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric ATP-releasing guanine-oxidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer.

Ablation of cardiac TIGAR preserves myocardial energetics and cardiac function in the pressure overload heart failure model.

Despite the advances in medical therapy, the morbidity and mortality of heart failure (HF) remain unacceptably high. HF results from reduced metabolism-contraction coupling efficiency, so the modulation of cardiac metabolism may be an effective strategy for therapeutic interventions. Tumor suppressor p53 (TP53) and its downstream target TP53-induced glycolysis and apoptosis regulator (TIGAR) are known to modulate cardiac metabolism and cell fate. To investigate TIGAR's function in HF, we compared myocardial, metabolic, and functional outcomes between TIGAR knockout (TIGAR-/-) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR-/- mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared with those of TIGAR-/- mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR-/- hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/Tg(Myh6-cre/Esr1) mice). The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR-targeted therapy to increase glucose metabolism will be a novel strategy for HF. NEW & NOTEWORTHY The present study is the first to demonstrate that TP53-induced glycolysis and apoptosis regulator (TIGAR) is upregulated in the myocardium during experimental heart failure (HF) in mice and that TIGAR knockout can preserve the heart function and myocardial energetics during HF. Reducing TIGAR to enhance myocardial glycolytic energy production is a promising therapeutic strategy for HF.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

BACKGROUND: Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. METHODS: Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. RESULTS: MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. CONCLUSIONS: MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

A distinctive patchy osteomalacia characterises Phospho1-deficient mice.

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.

Increased Resistance to Intradermal Francisella tularensis LVS Infection by Inactivation of the Sts Phosphatases.

The Suppressor of TCR signaling proteins (Sts-1 and Sts-2) are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic lineages, including T lymphocytes. Mice lacking Sts expression are characterized by enhanced T cell responses. Additionally, a recent study demonstrated that Sts-/- mice are profoundly resistant to systemic infection by Candida albicans, with resistance characterized by enhanced survival, more rapid fungal clearance in key peripheral organs, and an altered inflammatory response. To investigate the role of Sts in the primary host response to infection by a bacterial pathogen, we evaluated the response of Sts-/- mice to infection by a Gram-negative bacterial pathogen. Francisella tularensis is a facultative bacterial pathogen that replicates intracellularly within a variety of cell types and is the causative agent of tularemia. Francisella infections are characterized by a delayed immune response, followed by an intense inflammatory reaction that causes widespread tissue damage and septic shock. Herein, we demonstrate that mice lacking Sts expression are significantly resistant to infection by the live vaccine strain (LVS) of F. tularensis Resistance is characterized by reduced lethality following high-dose intradermal infection, an altered cytokine response in the spleen, and enhanced bacterial clearance in multiple peripheral organs. Sts-/- bone marrow-derived monocytes and neutrophils, infected with F. tularensis LVS ex vivo, display enhanced restriction of intracellular bacteria. These observations suggest the Sts proteins play an important regulatory role in the host response to bacterial infection, and they underscore a role for Sts in regulating functionally relevant immune response pathways.

Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

Vitamin B6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B6 vitamers were detected as compared with the control.

INPP5E regulates phosphoinositide-dependent cilia transition zone function.

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2'-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2'-deoxyguanosine.

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4-phosphate in breast cancer.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIß in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis.

Depletion of Inositol Polyphosphate 4-Phosphatase II Suppresses Callosal Axon Formation in the Developing Mice.

The corpus callosum is a bundle of nerve fibers that connects the two cerebral hemispheres and is essential for coordinated transmission of information between them. Disruption of early stages of callosal development can cause agenesis of the corpus callosum (AgCC), including both complete and partial callosal absence, causing mild to severe cognitive impairment. Despite extensive studies, the etiology of AgCC remains to be clarified due to the complicated mechanism involved in generating AgCC. The biological function of PI3K signaling including phosphatidylinositol-3,4,5-trisphosphate is well established in diverse biochemical processes including axon and dendrite morphogenesis, but the function of the closely related phosphatidylinositol-3,4,-bisphosphate (PI(3,4)P2) signaling, particularly in the nervous system, is largely unknown. Here, we provide the first report on the role of inositol polyphosphate 4-phosphatase II (INPP4B), a PI(3,4)P2 metabolizing 4-phosphatase in the regulation of callosal axon formation. Depleting INPP4B by in utero electroporation suppressed medially directed callosal axon formation. Moreover, depletion of INPP4B significantly attenuated formation of Satb2-positive pyramidal neurons and axon polarization in cortical neurons during cortical development. Taken together, these data suggest that INPP4B plays a role in the regulating callosal axon formation by controlling axon polarization and the Satb2-positive pyramidal neuron population. Dysregulation of INPP4B during cortical development may be implicated in the generation of partial AgCC.

Specific behavioural phenotype and secondary cognitive decline as a result of an 8.6 Mb deletion of 2q32.2q33.1.

Chromosomal abnormalities involving 2q32q33 deletions are very rare and present with a specific phenotype. This case report describes a 37-year-old female patient with 2q32q33 microdeletion syndrome presenting with the characteristic features, but with the addition of secondary cognitive decline. Molecular karyotyping was performed on the patient and her parents. It revealed an 8.6 megabase deletion with the proximal breakpoint in the chromosome band 2q32.2 and the distal breakpoint in 2q33.1. The deletion encompassed 22 known genes, including theGLS,MYO1B,TMEFF2,PGAP1andSATB2genes. The observed deletion was confirmed using a paralogue ratio test. This case report provides further evidence that theSATB2gene, together withGLS,MYO1B,TMEFF2and possiblyPGAP1,is a crucial gene in 2q32q33 microdeletion syndrome. TheSATB2gene seems to be crucial for the behavioural problems noted in our case, but deletion of theGLS,MYO1BandTMEFF2genes presumably contributed to the more complex behavioural characteristics observed. Our patient is also, to our knowledge, the only patient with 2q32q33 microdeletion syndrome with secondary cognitive decline.

A phosphoinositide conversion mechanism for exit from endosomes.

Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.