Dissecting inositol pyrophosphates signalling

Lead Research Organisation: MRC Cell Biology Unit

Abstract

My research is focused on the study of inositol phosphates, a large family of small molecules that regulate many essential and basic functions in all living organisms, including heart muscle contraction, the release of neurotransmitters in the brain, mammalian cell survival and the migration of cells of the immune system. Two new members of this family (called IP7 and IP8) have the exceptionally unusual property of containing high energy Pyro-phosphate bonds. This is similar to the high energy phosphate bond found in ATP, the main energy storage molecule found in all cells. Like ATP, IP7 and IP8 are also found in all cells analyzed so far, ranging from fungi to humans, but very little is known about their physiological functions. The release of energy from their high energy bonds could, potentially, drive a variety of fundamental biochemical processes and the goal of my research is to identify the physiological role of this novel and exciting class of high-energy molecules.

Technical Summary

Inositol phosphates (IP) occur ubiquitously in biological tissues. The most widely studied IP is inositol 1,4,5-trisphosphate (IP3), the second messenger that triggers calcium release from intracellular stores. Interestingly, the six carbons that comprise the fully phosphorylated inositol ring of IP6 (Inositol hexakisphosphate, also known as phytic acid) can be further phosphorylated, creating more polar inositol polyphosphates. These higher inositol phosphates contain one, two or more high-energy Pyro-phosphate bonds, similar to the high-energy bonds present in ATP. The best-characterized inositol pyrophosphates are the diphosphoinositol pentakisphosphate (IP7) and the bis-diphosphoinositol tetrakisphosphate (IP8). In cells the IP6 concentration is very stable, while IP7 and IP8 are metabolized at a very high rate. Every hour, 20 to 50% of the large IP6 reservoir cycles through inositol pyrophosphates, and it has been suggested that this cycle represent a molecular switch activity that regulates intracellular signalling.||Our ability to produce radiolabeled IP7 with high specific activity has allowed us to detect the direct transfer of the radiolabel beta phosphate from IP7 to proteins. This phosphorylation mechanism defines a yet new kind of protein post-translational modification. A recent study has shown that inositol pyrophosphates donate the phosphate group to pre-phosphorylated serines, thus generating a pyro-phosphorylated protein. Although we are able to reproducibly transfer phosphate moieties from IP7 to pre-phosphorylated serine of proteins in vitro and have indirect evidence that IP7 can directly mediate phosphorylation in vivo, the molecular mechanisms underlying IP7-dependent protein pyro-phosphorylation in vivo and the physiological consequence of such, are not fully elucidated yet. The main objective of my laboratory is to identify the physiological functions of inositol pyro-phosphates. We are employing several experimental models from yeast Saccharomyces cerevisiae, a simple but powerful genetic model, to mouse knockout to mimic human physiology. We are routinely using several biochemical, molecular biology and cell biology techniques.||It has become increasingly clear that inositol pyrophosphates have a very basic signalling function inside all eukaryotic cells. It is likely that the inositol pyro-phosphates have evolved dedicated roles due to their unique biochemistry. The potential therapeutic potential of such research is supported by the recent identification of the critical role inositol pyrophosphates in the regulation of insulin secretion and oncogenic processes. We have substantially contributed to both studies.

Publications

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Azevedo C (2016) The new world of inorganic polyphosphates. in Biochemical Society transactions

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Azevedo C (2014) Functions of inorganic polyphosphates in eukaryotic cells: a coat of many colours. in Biochemical Society transactions

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Azevedo C (2011) The signaling role of inositol hexakisphosphate kinases (IP6Ks). in Advances in enzyme regulation

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Azevedo C (2017) Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective. in Trends in biochemical sciences

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Azevedo C (2010) Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1). in Methods in molecular biology (Clifton, N.J.)

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Azevedo C (2009) Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. in Proceedings of the National Academy of Sciences of the United States of America

 
Title LMCB Recruitment Poster 
Description A postdoc in my laboratory, Miranda Wilson, designed a poster that was chosen to advertise the LMCB's PhD programme. This poster will be sent to institutions around Europe for display. 
Type Of Art Artwork 
Year Produced 2014 
Impact Our institute will now be more competitive in attracting high quality students from UK and abroad. 
 
Description EC Marie Curie Re-integrationFellowship
Amount £80,000 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 01/2006 
End 01/2008
 
Description HFSP-Grant Agreement: RGP00048/2009-C/Human Frontiers Science Program
Amount £225,000 (GBP)
Funding ID RGP00048/2009-C 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 09/2009 
End 08/2013
 
Description Marie Curie Intra-European Fellowships
Amount € 195,454 (EUR)
Funding ID EU project 752903 - PHEMDD - GAP-752903 
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 07/2017 
End 07/2019
 
Title PAGE to resolve inositol polyphosphates 
Description I develop a new method to separate and differentially detect inositol polyphosphate using gel electrophoresis. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact This new methods has enormously facilitate inositol pyrophosphates research. Can be use to identify IP6K inhibitors. Although this technology was just published in May 2009 by October 2009 the majority of scientists working on inositol pyrophosphate research have adopted this new method. I hosted several students and postdoc from around the world in my lab to directly learn this powerful technology. 
URL https://www.jove.com/video/3027/preparation-of-quality-inositol-pyrophosphates
 
Title Ti02 purification of inositol phosphates 
Description New purification procedure to extract inositol phosphate from cells 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact One published paper describing this new technology A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine. Wilson MS, Bulley SJ, Pisani F, Irvine RF, Saiardi A. Open Biol. 2015 Mar;5(3):150014. Many paper are coming out using this methods that have helped to develop new chemical tools, such: Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate. Pavlovic I, Thakor DT, Bigler L, Wilson MS, Laha D, Schaaf G, Saiardi A, Jessen HJ. Angew Chem Int Ed Engl. 2015 Aug 10;54(33):9622-6. 
 
Description ACR : IPMK Functional studies 
Organisation University of Pennsylvania
Department Perelman School of Medicine
Country United States 
Sector Academic/University 
PI Contribution Reagents and discussion
Collaborator Contribution Reagents and discussion and ideas
Impact WE publish two reviews, one on PubMed 17981594, the second is an important book chapter in Wiley Encyclopedia of Chemical Biology. this year we publish a basic science manuscript PubMed 21220345.
Start Year 2006
 
Description AR : Analysis of inositol phosphates signaling in neuronal cells. 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution We provided the technical and theoretical expertise to analyze inositol phosphates.
Collaborator Contribution They have allowed me to study signaling by inositol phosphates in neuronal cell. Neurons are an excellent experimental system to study signaling mechanism that due to technical difficulty is to often misuse or not use. Thanks to this collaboration I now using neuronal cell as experimental model.
Impact This has been a very productive collaboration that generated high profile publication (Nature Neuroscience) PubMed 20118926 and (Molecular Biology of the Cell) PMID:23864704
Start Year 2006
 
Description CL : Inositides distribution and quantification using Multi-isotope Imaging Mass Spectrometry. 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution To this project I supply reagents, yeast strains and inositol signaling expertise.
Collaborator Contribution This partnership opens the unique possibility to direct analyze-imagining inositol inside cells. Claude with his expertise and technology will allow unique investigation.
Impact This collaboration has allowed me to win a very prestigious HFSP grant thus enhancing (economically and scientifically) the profile of the lab. At the moment the only outputs this collaboration generated is extra money and higher scientific profile for the lab. We generate one publication as reported in my papers output
Start Year 2009
 
Description Developing inositol phosphate chemical tools 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Developing inositol phosphate chemical tools
Collaborator Contribution organic synthesis
Impact One publication Pavlovic I, Thakor DT, Bigler L, Wilson MS, Laha D, Schaaf G, Saiardi A, Jessen HJ. Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate. Angew Chem Int Ed Engl. 2015 Aug 10;54(33):9622-6. PMID: 26014370.
Start Year 2013
 
Description EN : Control of Autophagy by inositol pyrophosphates. 
Organisation Tokai University
Department School of Medicine
Country Japan 
Sector Academic/University 
PI Contribution Data analysis and reagent exchange.
Collaborator Contribution Eiichiro provided the lab with unique data and reagents
Impact Manuscript PMID: 20883817 Manuscript PMID: 21652713
Start Year 2006
 
Description Inositol phosphate and phosphate homeostasis in Dictyostelium discoideum 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We developed Dictyostelium discoideum as a model to study inositol phosphate metabolism and the inorganic polyphosphate (polyP) role in biology. Our experience in inositol polyphosphate and polyP analysis have rapidly facilitated the development of this model thanks to the help of our collaborator. We are now using amoeba to understand the role of the cited molecules to control cellular phosphate homeostasis.
Collaborator Contribution He transferred his know-how on Amoeba biology to my laboratory personnel, furthermore, he has given us tools and reagents essential to initiate to perform Dictyostelium discoideum research.
Impact This collaboration generated two scientific publications: 1: Livermore TM, Chubb JR, Saiardi A. Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum. Proc Natl Acad Sci U S A. 2016 Jan 26;113(4):996-1001. 2: Pisani F, Livermore T, Rose G, Chubb JR, Gaspari M, Saiardi A. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis. PLoS One. 2014 Jan 9;9(1):e85533. More importantly a postdoc of the laboratory proposing a project based on this collaboration was awarded a Marie Curie Fellowship (EU project 752903 - PHEMDD - GAP-752903)
Start Year 2013
 
Description Inositol pyrophosphate signaling in the yeast S. pombe 
Organisation Heinrich Heine University Düsseldorf
Country Germany 
Sector Academic/University 
PI Contribution Transfer of my inositol pyrophosphate and inorganic polyphosphate (polyP) theoretical and practical know-how. Biochemical analysis of Schizosaccharomyces pombe inositol kinases mutant strains.
Collaborator Contribution Initiated the project identifying by genetic studies key roles for inositol phosphate kinases of Schizosaccharomyces pombe, Is providing stains, expertise, tools and genetic analyses.
Impact This collaboration is just started, thus there are not output to be listed. However, a important publication has been just been accepted (Mol Cell Biol. 2018 Feb 12. pii: MCB.00047-18. doi: 10.1128/MCB.00047-18.) in wich we describe the importance of the phosphatase domain of PP-IP5K to regulate the level of inositol pyrophosphates. This collaboration is multidisciplinary, involving a yeast geneticist and a biochemist.
Start Year 2017
 
Description Roles of Inositol Phosphates in Viral Biology 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our inositol phosphate analysis technologies allow us to analyse the inositol phosphate levels in HIV virion. We are also theoretically contributing to this collaborative project by transferring our unique inositol metabolic pathways know-own.
Collaborator Contribution The partner is a HIV structural biologist and bring in this collaboration the HIV biology expertise
Impact This collaboration is multidisciplinary, involving a structural biologist/ immunologist and a biochemist. This collaboration generated a published paper and a second manuscript just submitted. IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis. Mallery DL, et al. Elife. 2018 May 31;7. pii: e35335.
Start Year 2017
 
Description Studying inositol phosphate signalling in Cryptococcus neoformans 
Organisation University of Sydney
Country Australia 
Sector Academic/University 
PI Contribution I will analyse the inositol polyphosphate signal of Cryptococcus neoformans mutant generated in Dr. Julienne Djordjevic laboratory
Collaborator Contribution Dr J. Djordjevic is currently generating and characterising Cryptococcus neoformans mutant strains defective in inositol phosphate kinases
Impact This collaboration has been awarded a NHMRC research grant and I was CO-PI of Dr J. Djordjevic application.
Start Year 2013
 
Description Studying inositol phosphate signalling in Trypanosome 
Organisation University of Georgia
Country United States 
Sector Academic/University 
PI Contribution We analyse the biochemistry of Trypanosome mutant in the enzymes responsible for inositol pyrophosphate synthesis
Collaborator Contribution Generate the mutants and study the in vivo phenotype of altered inositides signalling
Impact We are co-organising a 2015 meeting sponsored by the Biochemical Society
Start Year 2013
 
Description plant inositol pyrophosphate 
Organisation Eberhard Karls University of Tubingen
Country Germany 
Sector Academic/University 
PI Contribution analysis of inositol phosphate levels excenge of reagent and tecnologies
Collaborator Contribution developing the research line and generating plant mutant lines
Impact One publication VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis. Laha D, Johnen P, Azevedo C, Dynowski M, Weiß M, Capolicchio S, Mao H, Iven T, Steenbergen M, Freyer M, Gaugler P, de Campos MK, Zheng N, Feussner I, Jessen HJ, Van Wees SC, Saiardi A, Schaaf G. Plant Cell. 2015 Apr;27(4):1082-97.
Start Year 2013
 
Title PAGE to resolve inositol polyphosphates 
Description New method to analyze inositol phosphates quality, quantity and kinase reactions using gel electrophoresis. 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed No
Impact Signed confidentiality disclosure agreement between MRC and Echelon Biosciences Incorporated (Salt Lake City, UT)
 
Description 2013 MRC Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We have divulgate our science to the general public on the MRC open day (29 June 2013) about ~200 peoples (general public) maily family with cildren visited our stand

It was rewording for us and instructive and fun for the audience. Likely this type of event will be yearly repeated.
Year(s) Of Engagement Activity 2013
 
Description Inositol pyrophosphate in cell biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presenting my work experience at the following meeting: "Cosentini illustri" Convegno Cosenza 2009; 18-20 February 2008, Cosenza Italy

I further realize that in Italy science don't have any chance.
Year(s) Of Engagement Activity 2008
 
Description Interview for national news on immortality 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact The radio programme discussed life after death. Dr. Miranda Wilson (Postdoc of the laboratory) was interviewed for a section on HeLa and other cell lines being a form of immortality.
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/programmes/w3csv3f3
 
Description TEDx theme Brain Drain (23 February 2017, Cosenza, Italy) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The different presentation focused on the Brain Drain that Italy is witnessing in recent years. I presented my experience and outline the difference approach to science and to scientist that exist in different part of the world. I also discuss the concept that "Science is not a democracy" something that the general public found difficult to accept.
Year(s) Of Engagement Activity 2017
URL https://www.ted.com/tedx/events/19418
 
Description UNISTEM Day (U.Calabria, Italy) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Speaker to the 2014 Unistem Day, A stem cell event dedicated to high school students University of Calabria (Italy) 15 March 2014
Year(s) Of Engagement Activity 2014
URL http://users2.unimi.it/unistem/index.php/archive-unistemday/?lang=en