Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes

Conway, Stuart J.; Gardiner, James; Grove, Simon J. A.; Johns, Melloney K.; Lim, Ze-Yi; Painter, Gavin F.; Robinson, Danielle; Schieber, Christine; Thuring, Jan Willem; Wong, Leon S.‐M.; Yin, Meng‐Xin; Burgess, Antony W.; Catimel, Bruno; Hawkins, Phillip T.; Ktistakis, Nicholas T.; Stephens, Len R.; Holmes, Andrew B.

doi:10.1039/b913399b

Cited by 56 publications

(73 citation statements)

References 88 publications

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“…This intermediate was reported by Holmes and co-workers, and can be obtained from myo -inositol in two steps. [20] Along this strategy, allylation of 3 , followed by acetal cleavage, formed allyl ether 4 in 84% yield. para -Methoxybenzyl (PMB) protection of the hydroxyl groups and removal of the allyl protecting group provided reliable and scalable synthetic access to alcohol 5 , which was then converted into the bisphosphonate-containing intermediate 6 .…”

Section: Resultsmentioning

confidence: 99%

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

Hager

Wang

et al. 2016

Chemistry A European J

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The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.

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Section: Resultsmentioning

confidence: 99%

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

Hager

Wang

et al. 2016

Chemistry A European J

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“…6 and 5PP-InsP 5 Affinity Reagents. Affinity-based probes have been widely used by the inositol community, in particular, to identify interactions between phosphatidyl inositols and their protein targets (27)(28)(29)(30)(31). Furthermore, immobilized InsP 6 has enabled the identification of two enzymes involved in InsP biosynthesis (12,32,33) and allowed for affinity capture of the Ku protein, a required factor for nonhomologous end joining (34).…”

Section: Significancementioning

confidence: 99%

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms

Chong

Perlman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

116

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Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.inositol pyrophosphates | affinity reagents | protein pyrophosphorylation | signal transduction | metabolism S ignal transduction pathways and metabolic circuits are essential for cell homeostasis and survival. These two types of networks have historically been viewed as separate entities, but it is becoming increasingly clear that they must be coordinately regulated. Indeed, growth factor-stimulated signaling pathways can promote the metabolic activity of the cell (1). Conversely, the activity of signaling proteins can be controlled by specific metabolites (2), either by allosteric mechanisms or via nutrient-sensitive covalent modifications, such as acetylation (3) and glycosylation (4).The highly phosphorylated inositol polyphosphates (InsPs), and in particular the inositol pyrophosphates (PP-InsPs), are primed to provide additional junctures between signaling and metabolic networks (5-7). A cascade of phosphorylation reactions converts the secondary messenger inositol trisphosphate (InsP 3 ) to the fully phosphorylated inositol hexakisphosphate (InsP 6 ) (8). Subsequent action of inositol hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks) furnishes the PP-InsP messengers, a unique class of signaling molecules containing one or two high-energy phosphoanhydride bonds (Fig. 1A) (6,7,9). A number of studies have indicated a central role for PP-InsPs in metabolic reprogramming and phosphorylation-based signaling at the cellular and organismal level. For example, the biochemical properties of the IP6Ks confer an "energy sensing" function onto these enzymes (10). Because the IP6Ks have a K m for ATP between 1.0 and 1.4 mM-concentrations that are similar to the intracellular ATP levels-t...

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“…Using a panel of chemically synthesised lipids covalently coupled to a solid support, which we have extensively characterised and validated in our lab (Conway et al, 2010;Delon et al, 2004;Krugmann et al, 2002;Manifava et al, 2001;Ridley et al, 2001), we tested lysates from the clonal cell line expressing the wt GFP-Atg13 for binding to PtdIns3P, PtdIns4P, PtdIns(3,4)P 2 , PtdIns(3,5)P 2 , PtdIns(3,4,5)P 3 , phosphatidic acid (PA), diacylglycerol (DAG), cardiolipin and sphingosine. Under conditions where endogenous Atg16 did not bind to any of the beads tested, Atg13 bound to PA-, PtdIns3P-and PtdIns4P-coated beads and more weakly to those coated with PtdIns(3,4,5)P 3 (Fig.…”

Section: A Site On Atg13 Is Important For Lipid Binding and Translocamentioning

confidence: 99%

Dynamic association of the ULK1 complex with omegasomes during autophagy induction

Karanasios

Stapleton

Manifava

et al. 2013

Journal of Cell Science

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215

255

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SummaryInduction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

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Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes

Cited by 56 publications

References 88 publications

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms

Dynamic association of the ULK1 complex with omegasomes during autophagy induction

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