Inositol pyrophosphates are cellular signals that are created by the actions of inositol kinases and are degraded by highly active inositol phosphatases. The potent actions of these phosphatases ...suggest these signals must be created near their sites of action. To identify sites where the inositol kinase, PPIP5K1 acts, we performed affinity purification of PPIP5K1 from HEK293 cells and analyzed these samples using mass spectrometry to identify the proteins pesent (10.1016/j.cellsig.2016.02.002) 1. We further decreased PPIP5K1 levels in HeLa cells and treated these with PPIP5K1 siRNA. We then monitored the motility of these cells in Scratch assays.
Inhibition of carbonic anhydrase IX in glioblastoma multiforme Amiri, Abdolali; Le, Phuong Uyen; Moquin, Alexandre ...
European journal of pharmaceutics and biopharmaceutics,
December 2016, 2016-Dec, 2016-12-00, 20161201, Letnik:
109
Journal Article
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Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among ...gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.
Cellular signaling involves coordinated regulation of many events. Scaffolding proteins are crucial regulators of cellular signaling, because they are able to affect numerous events by coordinating ...specific interactions among multiple protein partners in the same pathway. Scaffolding proteins often contain intrinsically disordered regions (IDR) that facilitate the formation and function of distinct protein complexes. We show that PPIP5K1 contains an unusually long and evolutionarily conserved IDR. To investigate the biological role(s) of this domain, we identified interacting proteins using affinity purification coupled with mass spectrometry. Here, we report that PPIP5K1 is associated with a network of proteins that regulate vesicle-mediated transport. We further identified exocyst complex component 1 as a direct interactor with the IDR of PPIP5K1. Additionally, we report that knockdown of PPIP5K1 decreases motility of HeLa cells in a wound-healing assay. These results suggest that PPIP5K1 might play an important role in regulating function of exocyst complex in establishing cellular polarity and directional migration of cells.
•PPIP5K1 contains a long intrinsically disordered C-terminal region.•The PPI5K IDR is evolutionarily conserved in higher eukaryotes.•PPIP5K1 associates with an array of proteins involved in vesicle-mediated transport and cytoskeletal modulators.•PPIP5K1 directly interacts with exocyst complex component 1 via its IDR.•Knocking down PPIP5K1 in HeLa cells significantly impacts cellular motility.
Aging is a highly complex, multifactorial process. We use the yeast
Saccharomyces cerevisiae as a model to study the mechanisms of cellular aging in multicellular eukaryotes. To address the inherent ...complexity of aging from a systems perspective and to build an integrative model of aging process, we investigated the effect of calorie restriction (CR), a low-calorie dietary regimen, on the metabolic history of chronologically aging yeast. We examined how CR influences the age-related dynamics of changes in the intracellular levels of numerous proteins and metabolites, carbohydrate and lipid metabolism, interorganellar metabolic flow, concentration of reactive oxygen species, mitochondrial morphology, essential oxidation–reduction processes in mitochondria, mitochondrial proteome, cardiolipin in the inner mitochondrial membrane, frequency of mitochondrial DNA mutations, dynamics of mitochondrial nucleoid, susceptibility to mitochondria-controlled apoptosis, and stress resistance. Based on the comparison of the metabolic histories of long-lived CR yeast and short-lived non-CR yeast, we propose that yeast define their long-term viability by designing a diet-specific pattern of metabolism and organelle dynamics prior to reproductive maturation. Thus, our data suggest that longevity in chronologically aging yeast is programmed by the level of metabolic capacity and organelle organization they developed, in a diet-specific fashion, prior to entry into a non-proliferative state.
Diphosphoinositol phosphates (diInsPs) are evolutionarily conserved high-energy inositol derivatives that have emerged as key regulators of fundamental cellular processes such as energy metabolism, ...genomic stability, vesicle transport, and apoptosis. The molecular mechanisms through which diInsPs mediate such a wide range of cellular processes are poorly understood. In this thesis, we focus on PPIP5K1, which catalyzes the addition of a âLphosphate bond to position 1 of a fully phosphorylated inositol ring. This enzyme was initially characterized as containing dual kinase/phosphatase domains, but its cellular functions have not been well characterized. Our overall goal was to characterize PPIP5K1 structure and function in order to identify how it generates diInsP products, how it is regulated, and how it may regulate distinct cellular processes. We have identified a previously unrecognized intrinsically disordered region (IDR) in the C terminal domain of PPIP5K1 and shown that it is a conserved feature of PPIP5K isoforms across higher eukaryotes. We therefore have reassigned the predicted domain composition of PPIP5K1 and subsequently used a proteomics approach to identify a scaffolding role for this IDR. We next generated a protein interaction network for PPIP5K1, which revealed a previously unknown involvement in vesicle trafficking through direct interactions of PPIP5K1 with the vesicle tethering exocyst complex. We also tested PPIP5K1 for its impact on cell survival, as had previously been reported for diInsPs generated by the IP6K family of enzymes. We discovered that overexpression of PPIP5K1 in mammalian cells protected them from genotoxic stresses, while overexpression of IP6K2, consistent with previous reports, increases cell sensitivity to these same agents. We investigated the cellular mechanisms underlying the anti-apoptotic actions of PPIP5K1 and discovered that PPIP5K1 reduces activation of pro-apoptotic p53 and death receptor pathways, while upregulating and activating anti-apoptotic proteins such as p21 and p27. Therefore, PPIP5K1 and IP6K2, which generate different InsP7 molecules and cooperate to form InsP8, have opposite impacts on cell viability.
Inositol hexakisphosphate kinase 2 (IP6K2) potentiates pro-apoptotic signalling and increases the sensitivity of mammalian cells to cytotoxic agents. Diphosphoinositol pentakisphosphate kinase ...(PPIP5K) generates inositol pyrophosphates (InsPPs) that are structurally distinct from those produced by IP6K2 and their possible roles in affecting cell viability remain unclear. In the present study, we tested the impact of PPIP5K1 on cellular sensitivity to various genotoxic agents to determine if PPIP5K1 and IP6K2 contribute similarly to apoptosis. We observed that PPIP5K1 overexpression decreased sensitivity of cells toward several cytotoxic agents, including etoposide, cisplatin, and sulindac. We further tested the impact of PPIP5K1 overexpression on an array of apoptosis markers and observed that PPIP5K1 decreased p53 phosphorylation on key residues, including Ser-15, -46, and -392. Overexpression of a kinase-impaired PPIP5K1 mutant failed to protect cells from apoptosis, indicating this protection is a consequence PPIP5K1 catalytic activity, in contrast with the sensitivity conferred by IP6K2, which is dependent on both catalytic and non-catalytic functions. These observations reveal distinct roles for PPIP5K1 and IP6K2 and the InsPPs they produce in controlling cell death.
Growing evidence supports the view that LDs (lipid droplets) are dynamic organelles that can serve both as an intracellular signalling compartment and as an organizing platform orchestrating many ...vital processes in eukaryotic cells. It has become clear that the LDs-confined deposition and lipolytic degradation of neutral lipids define longevity in multicellular eukaryotic organisms and yeast. We summarize the evidence in support of the essential role that LDs play in longevity regulation and propose several molecular mechanisms by which these dynamic organellar compartments control the aging process in multicellular eukaryotes and yeast.
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