Protocols for Regulation and Study of Diphosphoinositol Polyphosphates

Abstract

The roles of diphosphoinositol polyphosphates (DIPs) in mammalian cell biology have been difficult to determine because of the lack of tools known to regulate their levels. I have determined a series of protocols that regulate these DIPs, and these can be used to further our understanding of these molecules. Sorbitol and sucrose significantly raised levels of bis-diphosphoinositol tetrakisphosphate ([PP] 2 -InsP 4 ) but slightly lowered levels of diphosphoinositol pentakisphosphate (PP-InsP 5 ) in DDT 1 MF-2 cells. These effects correlate with the ability of hyperosmotic stress to interfere with protein trafficking described previously and suggest that [PP] 2 -InsP 4 specifically impedes protein trafficking. The effects on [PP] 2 -InsP 4 were not regulated by extracellular signal-regulated kinase or phospholipase D, as exemplified by the lack of effect of U0126 and butan-1-ol. I have also found that genistein potently and rapidly lowers levels of [PP] 2 -InsP 4 , whereas a similar inhibitor, herbimycin, was without effect. Thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca 2ϩ -ATPase pump inhibitor previously shown to selectively lower PP-InsP 5 after short-term treatment, also selectively raises PP-InsP 5 after a longer treatment. The calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine significantly lowered all higher inositol phosphates, as well as DIPs, whereas the calmodulin-dependent kinase inhibitors methyl 9-(S)-12- ,6]benzodiazocine-2,3,9,10,11,12-hexahydro-10-(R)hydroxy-9-methyl-1-oxo-10-carboxylate (K-252a) and 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) were without effect. W-7 and chlorpromazine also lowered levels of phosphatidylinositol 4,5-bisphosphate and ATP but greatly increased levels of phosphatidylinositol 4-phosphate. Trypan blue exclusion deemed that these doses were not cytotoxic. These results identify an increasing number of reagents that regulate DIP levels. Using these tools, and those described previously, we can further understand the roles of the DIPs in cell biology.

The Nudix hydrolase superfamily

“…This might be an aspect of normal cofactor regulation or it could be a means of eliminating non-functional, damaged nucleotides produced in the oxidising environment of the peroxisomal lumen, particularly under conditions of stress, when ROS production may increase. [110,111] and in non-enzymic protein phosphorylation [112]. A direct involvement of Ddp1p in vesicle trafficking is supported by its reported interaction with Yif1p, an integral membrane protein required for the fusion of endoplasmic reticulum (ER)-derived COPII transport vesicles with the Golgi [113], but its overall role in regulating the levels of DIPs and the possible interplay between these and p n A/Ap n A have not been adequately studied.…”

Inositol pyrophosphates: metabolism and signaling

“…Similarly, in primary hepatocytes the pool of IP 7 is turned over 10 times every 40 min, compared with 1/10th of the pools of IP 6 [13]. In mammalian cells, IP 8 levels are sensitive to a variety of pharmacological agents [28]. A single study on the variations of inositol polyphosphates with respect to the cell cycle in WRK-1 mammalian cell has shown a twofold difference in IP 7 levels between the G1 and S phases of the cell cycle [29].…”

Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis

“…Long before the isolation or cloning of the second inositol pyrophosphate synthesizing enzyme PPIP5K (Choi et al , 2007 ;Fridy et al , 2007 ), which is indispensable for the formation of 1,5-PP 2 -InsP 4 (Figure 2), Safrany (2004) identifi ed a range of conditions and drugs that affect DPIP levels among them hyperosmotic conditions and drugs also lowering cellular ATP levels (Safrany , 2004 ). At the same time, the Shears laboratory, searching for signaling conditions stimulating the biosynthesis of DPIPs, described a stimulation of PP 2 -InsP 4 levels by hyperosmotic stress (Pesesse et al , 2004 ).…”