Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases

BACKGROUND: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised. RESULTS: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (k(cat)/K(m) = 11, 8 and 16 × 10(3)M(-1)s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (k(cat)/K(m) = 13, 14 and 11 × 10(3)M(-1)s(-1) respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell. CONCLUSIONS: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap(5)A and Ap(6)A act as intracellular messengers

Simple ammonium salts acting on sigma-1 receptors yield potential treatments for cancer and depression

Sigma-1 and sigma-2 receptors are emerging therapeutic targets. We have identified that simple ammonium salts bind to these receptors and are effective in vivo. Radioligand binding assays were used to obtain structure-activity relationships of these salts. MTS assays were performed to determine their effect on growth in MCF7 and MDA-MB-486 cells. Anticancer properties were tested in NMRI mice transplanted with a fragment of mouse adenocarcinoma (MAC13). Antidepressant activity was tested using the forced-swim test and tailsuspension tests. Dipentylammonium (Ki 43 nM),tripentylammonium (Ki 15 nM) and trihexylammonium (Ki 9 nM) showed high affinity for the sigma-1receptor. Dioctanoylammonium had the highest affinity (K50 0.05 nM); this also showed the highest affinity for sigma-2 receptors (Ki 13 nM). Dipentylammonium was found to have antidepressant activity in vivo. Branched-chain ammonium salts showed lower affinity. Bis(2-ethylhexyl)ammonium (K50 29 μM), triisopentylammonium (K50 196 μM) and dioctanoylammonium showed a low Hill slope,and fitted a 2-site binding model for the sigma-1 receptor. We propose this two-site binding can be used to biochemically define a sigma-1 receptor antagonist. Bis(2-ethylhexyl)ammonium and triisopentylammonium were able to inhibit the growth of tumours in vivo. Cheap, simple ammonium salts act as sigma-1 receptor agonists and antagonists in vivo and require further investigation

A novel mechanism for the anti-cancer activity of aspirin and salicylates

© 2016 The Authors. Published by Spandidos Publications. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3892/ijo.2019.4701Epidemiological studies indicate that long-term aspirin usage reduces the incidence of colorectal cancer (CRC) and may protect against other non-CRC associated adenocarcinomas, including oesophageal cancer. A number of hypotheses have been proposed with respect to the molecular action of aspirin and other non‑steroidal anti‑inflammatory drugs in cancer development. The mechanism by which aspirin exhibits toxicity to CRC has been previously investigated by synthesising novel analogues and derivatives of aspirin in an effort to identify functionally significant moieties. Herein, an early effect of aspirin and aspirin-like analogues against the SW480 CRC cell line was investigated, with a particular focus on critical molecules in the epidermal growth factor (EGF) pathway. The present authors proposed that aspirin, diaspirin and analogues, and diflunisal (a salicylic acid derivative) may rapidly perturb EGF and EGF receptor (EGFR) internalisation. Upon longer incubations, the diaspirins and thioaspirins may inhibit EGFR phosphorylation at Tyr1045 and Tyr1173. It was additionally demonstrated, using a qualitative approach, that EGF internalisation in the SW480 cell line may be directed to endosomes by fumaryldiaspirin using early endosome antigen 1 as an early endosomal marker and that EGF internalisation may also be perturbed in oesophageal cell lines, suggestive of an effect not only restricted to CRC cells. Taken together and in light of our previous findings that the aspirin-like analogues can affect cyclin D1 expression and nuclear factor-κB localisation, it was hypothesized that aspirin and aspirin analogues significantly and swiftly perturb the EGFR axis and that the protective activity of aspirin may in part be explained by perturbed EGFR internalisation and activation. These findings may also have implications in understanding the inhibitory effect of aspirin and salicylates on wound healing, given the critical role of EGF in the response to tissue trauma

A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). PIKfyve function is required for homeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the Amyloid Precursor Protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. Here we utilise this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP's intracellular domain (AICD) to the HIV TAT domain, a cell permeable peptide allowing proteins to penetrate cells. The resultant TAT-AICD fusion protein is cell permeable and triggers an increase of PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT-AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the APP intracellular domain activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease

The cytotoxicity and synergistic potential of aspirin and aspirin analogues towards oesophageal and colorectal cancer

Background: Oesophageal cancer (OC) is a deadly cancer because of its aggressive nature with survival rates that have barely improved in decades. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of OC. Methods: The toxicity of aspirin and aspirin derivatives to OC and a colorectal cancer (CRC) cell line were investigated in the presence and absence of platins. Results: The data in this study show the effects of a number of aspirin analogues and aspirin on OC cell lines that originally presented as squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues fumaryldiaspirin (PN517) and the benzoylsalicylates (PN524, PN528 and PN529), were observed to be more toxic against the OC cell lines than aspirin. Both quantitative and qualitative apoptosis experiments reveal that these compounds largely induce apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The OE21 (SSC) and OE33 (ADC) cell lines were more sensitive to the aspirin analogues compared to the Flo-1 cell line (ADC). A non-cancerous oesophageal primary cells NOK2101, was used to determine the specificity of the aspirin analogues and cytotoxicity assays revealed that analogues PN528 and PN529 were selectively toxic to cancer cell lines, whereas PN508, PN517 and PN524 also induced cell death in NOK2101. In combination index testing synergistic interactions of the most promising compounds, including aspirin, with cisplatin, oxaliplatin and carboplatin against the OE33 cell line and the SW480 CRC cell line were investigated. Compounds PN517 and PN524, and to a lesser extent PN528, synergised with cisplatin against OE33 cells. Cisplatin and oxaliplatin synergised with aspirin and PN517 when tested against the SW480 cell line. Conclusion: These findings indicate the potential and limitations of aspirin and aspirin analogues as chemotherapeutic agents against OC and CRC when combined with platins

Phosphoinositide signalling in a neuroblastoma cell line.

In the studies described in this thesis, the ability of muscarinic agonists to initiate phosphoinositide metabolism, subsequently leading to the mobilisation of Ca2+ from intracellular stores was examined in permeabilised human neuroblastoma SH-SY5Y cells. Muscarinic receptors, determined as being of the M3 subtype, were found to possess different levels of receptor reserve for the production of the second messenger, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and the mobilisation of Ca2+ in this permeabilised cell preparation. The effects of agonist pretreatment of intact cells with the muscarinic agonist carbachol has also been examined. Such pretreatment attenuated the ability of muscarinic agonists to elicit Ca2+ mobilisation in permeabilised cells. The rate of desensitisation was dependent on the dose of agonist used, the temperature at which pretreatment was performed and was affected by the extracellular Ca2+ concentration. The mechanism of such receptor-mediated desensitisation was studied. The structure-activity relationships of a number of inositol phosphates and inositol phosphate analogues have also been studied with regards their interactions with the Ca2+-mobilising Ins(l,4,5)P3 receptor, Ins(l,4,5)P3 5-phosphatase and Ins(l,4,5)P3 3-kinase. This work has identified partial agonists at the Ins(l,4,5)P3 receptor, an inhibitor of Ins(l,4,5)P3 3-kinase which interacts poorly with the Ins(l,4,5)P3 receptor, and a highly potent and selective inhibitor of Ins(l,4,5)P3 5-phosphatase. In conclusion, the results obtained characterise a permeabilised cell preparation in which receptor coupling to phospholipase C is maintained, leading to the formation of Ins(l,4,5)P3 and mobilisation of Ca2+ from intracellular stores, and identifies this as a useful model in which this coupling can be modulated by cell membrane-impermeant agents

Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA–PK cofactor InsP(6) inhibits Ku mobility

Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA–PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP(6)) is a potent cofactor for DNA–PK activity in NHEJ. Specifically, InsP(6) binds to the Ku component of DNA–PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP(6) on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku’s interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP(6) was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku–GFP, and this could be reversed by replenishing cells with InsP(6). By expressing deletion mutants of Ku–GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP(6) enhances Ku mobility through a discrete region of Ku70, and modulation of InsP(6) levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins