Chemical tools to investigate inositol pyrophosphate protein interactions

Die Inositol-Pyrophosphate (PP-InsPs) sind eine ubiquitäre Gruppe hochphosphorylierter eukaryotischer Signalmoleküle. Sie werden mit einer Vielzahl zentraler zellulärer Prozesse in Verbindung gebracht, doch fehlt oft ein detailliertes Verständnis der einzelnen Signalereignisse, was zum Teil auf einen Mangel an chemischen Werkzeugen zurückzuführen ist. Diese Arbeit beschreibt die chemische Synthese, Validierung und Anwendung von PP-InsP-Affinitätsreagenzien zur Identifizierung von Proteinbindungspartnern von Inositolhexakisphosphat (InsP6) und 5-Diphosphoinositol-Pentakisphosphat (5PP-InsP5), zwei wichtigen eukaryotischen Metaboliten. Die Affinitätsreagenzien wurden entwickelt, um InsP6 und ein metabolisch stabiles 5PP-InsP5-Analogon auf drei verschiedene Arten darzustellen. Die Anwendung dieser triplexierten Reagenzien auf Säugetier-Lysate lieferte einen ersten umfassenden Datensatz in HCT116- und HEK293T-Zellen. Die Interaktome wurden mittels quantitativer Proteomik annotiert und enthüllten Hunderte von potenziellen Proteinbindungspartnern. Die quantitative Analyse der InsP6- und 5PP-InsP5-bindenden Proteine zeigte Beispiele für hochspezifische Protein-Ligand-Interaktionen auf. Biochemische Untersuchungen ergaben, dass Inositol-5-Phosphatasen, PRPS1 und spezifische Phosphatidyl-Inositolphosphat-Kinasen potenziell unentdeckte Zielproteine von PP-InsPs sind. Darüber hinaus wurde durch die Entwicklung einer neuen Strategie der Myo-Inositol-Desymmetrisierung erstmals die Synthese eines Affinitätsreagens auf der Basis von 1,5-Bisdiphosphoinositol-Tetrakisphosphat (1,5(PP)2-InsP4) beschrieben. Die Affinitätsreagenzien und die proteomischen Datensätze stellen für die Gemeinschaft leistungsstarke Ressourcen dar, um künftige Untersuchungen zu den vielfältigen Signalmodalitäten von Inositolpyrophosphaten einzuleiten.Inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is often missing, which can partially be attributed to a lack of chemical tools. This thesis describes the chemical synthesis, validation and application of PP-InsP affinity reagents to identify protein binding partners of inositol hexakisphosphate (InsP6) and 5-diphosphoinositol pentakisphosphate (5PP-InsP5), two important eukaryotic metabolites. The affinity reagents were developed to display InsP6 and a metabolically stable 5PP-InsP5 analog in three different ways. Application of these triplexed reagents to mammalian lysates provided a first comprehensive data set in HCT116 and HEK293T cells. The interactomes were annotated using quantitative proteomics and uncovered hundreds of potential protein binding partners. Quantitative analysis of InsP6 versus 5PP-InsP5 binding proteins highlighted examples of highly specific protein-ligand interactions. Biochemical studies primed inositol 5-phosphatases, PRPS1 and specific phosphatidyl inositol phosphate kinases as potentially undiscovered targets of PP-InsPs. Moreover, by developing a novel strategy of myo-inositol desymmetrization, the synthesis of an affinity reagent based on 1,5-bisdiphosphoinositol tetrakisphosphate (1,5(PP)2-InsP4) was described for the first time. The affinity reagents and the proteomic data sets constitute powerful resources for the community, to help launching future investigations into the multiple signaling modalities of inositol pyrophosphates

Using Biotinylated <i>myo</i>-Inositol Hexakisphosphate to Investigate Inositol Pyrophosphate–Protein Interactions with Surface-Based Biosensors

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated myo-inositol hexakisphosphates and their application in surface plasmon resonance and grating-coupled interferometry assays, to enable the rapid identification, validation, and kinetic characterization of InsP- and PP-InsP-protein interactions

Determination of the Cannabinoid CB1 Receptor’s Positive Allosteric Modulator Binding Site through Mutagenesis Studies

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators

Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility

Structural evidence for visual arrestin priming via complexation of phosphoinositols

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail

Association of biocompatible peritoneal dialysis solutions with peritonitis risk, treatment, and outcomes

Background and objectivesThe effect of biocompatible peritoneal dialysis (PD) solutions on PD-related peritonitis is unclear. This study sought to evaluate the relationship between use of biocompatible solutions and the probability of occurrence or clinical outcomes of peritonitis.Design, setting, participants, & measurementsThe study included all incident Australian patients receiving PD between January 1, 2007, and December 31, 2010, using Australia and New Zealand Dialysis and Transplant Registry data. All multicompartment PD solutions of neutral pH were categorized as biocompatible solutions. The independent predictors of peritonitis and the use of biocompatible solutions were determined by multivariable, multilevel mixed-effects Poisson and logistic regression analysis, respectively. Sensitivity analyses, including propensity score matching, were performed.ResultsUse of biocompatible solutions gradually declined (from 7.5% in 2007 to 4.2% in 2010), with preferential use among smaller units and among younger patients without diabetes mellitus. Treatment with biocompatible solution was associated with significantly greater overall rate of peritonitis (0.67 versus 0.47 episode per patient-year; incidence rate ratio, 1.49; 95% confidence interval [CI], 1.19 to 1.89) and with shorter time to first peritonitis (hazard ratio [HR], 1.48; 95% CI, 1.17 to 1.87), a finding replicated in propensity score-matched cohorts (HR, 1.36; 95% CI, 1.09 to 1.71).ConclusionsIn an observational registry study, use of biocompatible PD solutions was associated with higher overall peritonitis rates and shorter time to first peritonitis. Further randomized studies adequately powered for a primary peritonitis outcome are warranted.Yeoungjee Cho, Sunil V. Badve, Carmel M. Hawley, Stephen P. McDonald, Fiona G. Brown, Neil Boudville, Kym M. Bannister, Philip A. Clayton, David W. Johnso

Effects of biocompatible versus standard fluid on peritoneal dialysis outcomes

The clinical benefits of using "biocompatible" neutral pH solutions containing low levels of glucose degradation products for peritoneal dialysis compared with standard solutions are uncertain. In this multicenter, open-label, parallel-group, randomized controlled trial, we randomly assigned 185 incident adult peritoneal dialysis patients with residual renal function to use either biocompatible or conventional solution for 2 years. The primary outcome measure was slope of renal function decline. Secondary outcome measures comprised time to anuria, fluid volume status, peritonitis-free survival, technique survival, patient survival, and adverse events. We did not detect a statistically significant difference in the rate of decline of renal function between the two groups as measured by the slopes of GFR: -0.22 and -0.28 ml/min per 1.73 m(2) per month (P=0.17) in the first year in the biocompatible and conventional groups, respectively, and, -0.09 and -0.10 ml/min per 1.73 m(2) per month (P=0.9) in the second year. The biocompatible group exhibited significantly longer times to anuria (P=0.009) and to the first peritonitis episode (P=0.01). This group also had fewer patients develop peritonitis (30% versus 49%) and had lower rates of peritonitis (0.30 versus 0.49 episodes per year, P=0.01). In conclusion, this trial does not support a role for biocompatible fluid in slowing the rate of GFR decline, but it does suggest that biocompatible fluid may delay the onset of anuria and reduce the incidence of peritonitis compared with conventional fluid in peritoneal dialysis.David W. Johnson, Fiona G. Brown, Margaret Clarke, Neil Boudville, Tony J. Elias, Marjorie W.Y. Foo, Bernard Jones, Hemant Kulkarni, Robyn Langham, Dwarakanathan Ranganathan, John Schollum, Michael Suranyi, Seng H. Tan and David Voss on behalf of the balANZ Trial Investigator