Has Inositol Played Any Role in the Origin of Life?

Phosphorus, as phosphate, plays a paramount role in biology. Since phosphate transfer reactions are an integral part of contemporary life, phosphate may have been incorporated into the initial molecules at the very beginning. To facilitate the studies into early phosphate utilization, we should look retrospectively to phosphate-rich molecules present in today's cells. Overlooked by origin of life studies until now, inositol and the inositol phosphates, of which some species possess more phosphate groups that carbon atoms, represent ideal molecules to consider in this context. The current sophisticated association of inositol with phosphate, and the roles that some inositol phosphates play in regulating cellular phosphate homeostasis, intriguingly suggest that inositol might have played some role in the prebiotic process of phosphate exploitation. Inositol can be synthesized abiotically and, unlike glucose or ribose, is chemically stable. This stability makes inositol the ideal candidate for the earliest organophosphate molecules, as primitive inositol phosphates. I also present arguments suggesting roles for some inositol phosphates in early chemical evolution events. Finally, the possible prebiotic synthesis of inositol pyrophosphates could have generated high-energy molecules to be utilized in primitive trans-phosphorylating processes

Protein pyrophosphorylation: moving forward

Genetic ablation of inositol pyrophosphate synthesis has established the fundamental importance of this class of molecules to the eukaryote cell. These studies, however, must be complemented by cell biology and biochemical approaches to appreciate the signalling involved in the processes regulated by inositol pyrophosphates. A recent study by Chanduri et al. published in the Biochemical Journal, by integrating multiple experimental approaches, demonstrated that inositol pyrophosphates regulate intracellular vesicular movement. In particular, the vesicular transport along the microtubule that is driven by the motor protein complex dynein. Importantly, one subunit of this cellular motor, dynein 1 intermediate chain 2, undergoes serine pyrophosphorylation, a post-translational modification driven by inositol pyrophosphates. The pyrophosphorylation status of this dynein intermediate chain regulates its interaction with dynactin, which recruits the motor to vesicles. This mechanistically might explain how inositol pyrophosphates control intracellular membrane trafficking. By dissecting the serine pyrophosphorylation process, this work increases our awareness of this modification, underappreciated by the scientific literature but probably not by the eukaryotic cell

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

Phosphate, as a cellular energy currency, essentially drives most biochemical reactions defining living organisms, and thus its homeostasis must be tightly regulated. Investigation into the role of inositol pyrophosphates (PP-IPs) has provided a novel perspective on the regulation of phosphate homeostasis. Recent data suggest that metabolic and signaling interplay between PP-IPs, ATP, and inorganic polyphosphate (polyP) influences and is influenced by cellular phosphate homeostasis. Different studies have demonstrated that the SPX protein domain is a key component of proteins involved in phosphate metabolism. How PP-IPs control some aspects of phosphate homeostasis has become clearer with the recently acquired crystal structures of SPX domains. We review here recent studies on eukaryote phosphate homeostasis and provide insights into future research

Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling

Inositol polyphosphates, in their water-soluble or lipid-bound forms, represent a large and multifaceted family of signalling molecules. Some inositol polyphosphates are well recognised as defining important signal transduction pathways, as in the case of the calcium release factor Ins(1,4,5)P3, generated by receptor activation-induced hydrolysis of the lipid PtdIns(4,5)P2 by phospholipase C. The birth of inositol polyphosphate research would not have occurred without the use of radioactive phosphate tracers that enabled the discovery of the “PI response”. Radioactive labels, mainly of phosphorus but also carbon and hydrogen (tritium), have been instrumental in the development of this research field and the establishment of the inositol polyphosphates as one of the most important networks of regulatory molecules present in eukaryotic cells. Advancements in microscopy and mass spectrometry and the development of colorimetric assays have facilitated inositol polyphosphate research, but have not eliminated the need for radioactive experimental approaches. In fact, such experiments have become easier with the cloning of the inositol polyphosphate kinases, enabling the systematic labelling of specific positions of the inositol ring with radioactive phosphate. This approach has been valuable for elucidating their metabolic pathways and identifying specific and novel functions for inositol polyphosphates. For example, the synthesis of radiolabelled inositol pyrophosphates has allowed the discovery of a new protein post-translational modification. Therefore, radioactive tracers have played and will continue to play an important role in dissecting the many complex aspects of inositol polyphosphate physiology. In this review we aim to highlight the historical importance of radioactivity in inositol polyphosphate research, as well as its modern usage

Inorganic polyphosphate in mammals: where's Wally?

Inorganic polyphosphate (polyP) is a ubiquitous polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. In prokaryotes and lower eukaryotes, both the presence of polyP and of the biosynthetic pathway that leads to its synthesis are well-documented. However, in mammals, polyP is more elusive. Firstly, the mammalian enzyme responsible for the synthesis of this linear biopolymer is unknown. Secondly, the low sensitivity and specificity of available polyP detection methods make it difficult to confidently ascertain polyP presence in mammalian cells, since in higher eukaryotes, polyP exists in lower amounts than in yeast or bacteria. Despite this, polyP has been given a remarkably large number of functions in mammals. In this review, we discuss some of the proposed functions of polyP in mammals, the limitations of the current detection methods and the urgent need to understand how this polymer is synthesized

Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate

The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic polyphosphate, since levels of both molecules increase at late stages of development. We detail here the methods used to extract inositol polyphosphates using perchloric acid and inorganic polyphosphate using acidic phenol. We also present the postextraction procedures to visualize and quantify these molecules by polyacrylamide gel electrophoresis and by malachite green assay

How inositol pyrophosphates control cellular phosphate homeostasis?

Phosphorus in his phosphate PO(4)(3-) configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control. Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production. Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate. Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link. In this review I will describe the recent literature on this topic, with in the hope of inspiring further research in this fascinating area of biology

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented

Inositol phosphate kinases in the eukaryote landscape

Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5-2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the 'inositol phosphate code' in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP6 (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3-3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology

The PPIP5K Family Member Asp1 Controls Inorganic Polyphosphate Metabolism in S. pombe

Inorganic polyphosphate (polyP) which is ubiquitously present in both prokaryotic and eukaryotic cells, consists of up to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this polymer is manifold and diverse and in fungi ranges from cell cycle control, phosphate homeostasis and virulence to post-translational protein modification. Control of polyP metabolism has been studied extensively in the budding yeast Saccharomyces cerevisiae. In this yeast, a specific class of inositol pyrophosphates (IPPs), named IP7, made by the IP6K family member Kcs1 regulate polyP synthesis by associating with the SPX domains of the vacuolar transporter chaperone (VTC) complex. To assess if this type of regulation was evolutionarily conserved, we determined the elements regulating polyP generation in the distantly related fission yeast Schizosaccharomyces pombe. Here, the VTC machinery is also essential for polyP generation. However, and in contrast to S. cerevisiae, a different IPP class generated by the bifunctional PPIP5K family member Asp1 control polyP metabolism. The analysis of Asp1 variant S. pombe strains revealed that cellular polyP levels directly correlate with Asp1-made IP8 levels, demonstrating a dose-dependent regulation. Thus, while the mechanism of polyP synthesis in yeasts is conserved, the IPP player regulating polyP metabolism is diverse