Rashna Bhandari

Inositol Pyrophosphate Synthesis by Inositol Hexakisphosphate Kinase 1 Is Required for Homologous Recombination Repair

Background: DNA repair by homologous recombination (HR) is critical to maintain genomic integrity. Results: Loss of inositol pyrophosphate synthesis by inositol hexakisphosphate kinase 1 (IP6K1) impairs HR in mammalian cells, leading to increased cell death. Conclusion: We report a new player in mammalian HR repair. Significance: As cancer chemotherapeutics act by causing DNA damage, IP6K1 inhibition may provide a novel route to supplement chemotherapy. Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are water-soluble inositol phosphates that contain high energy diphosphate moieties on the inositol ring. Inositol hexakisphosphate kinase 1 (IP6K1) participates in inositol pyrophosphate synthesis, converting inositol hexakisphosphate (IP6) to IP7. In the present study, we show that mouse embryonic fibroblasts (MEFs) lacking IP6K1 exhibit impaired DNA damage repair via homologous recombination (HR). IP6K1 knock-out MEFs show decreased viability and reduced recovery after induction of DNA damage by the replication stress inducer, hydroxyurea, or the radiomimetic antibiotic, neocarzinostatin. Cells lacking IP6K1 arrest after genotoxic stress, and markers associated with DNA repair are recruited to DNA damage sites, indicating that HR repair is initiated in these cells. However, repair does not proceed to completion because these markers persist as nuclear foci long after drug removal. A fraction of IP6K1-deficient MEFs continues to proliferate despite the persistence of DNA damage, rendering the cells more susceptible to chromosomal aberrations. Expression of catalytically active but not inactive IP6K1 can restore the repair process in knock-out MEFs, implying that inositol pyrophosphates are required for HR-mediated repair. Our study therefore highlights inositol pyrophosphates as novel small molecule regulators of HR signaling in mammals.

The emerging roles of inositol pyrophosphates in eukaryotic cell physiology

Inositol pyrophosphates are water soluble derivatives of inositol that contain pyrophosphate or diphosphate moieties in addition to monophosphates. The best characterised inositol pyrophosphates, are IP7 (diphosphoinositol pentakisphosphate or PP-IP5), and IP8 (bisdiphosphoinositol tetrakisphosphate or (PP)2-IP4). These energy-rich small molecules are present in all eukaryotic cells, from yeast to mammals, and are involved in a wide range of cellular functions including apoptosis, vesicle trafficking, DNA repair, osmoregulation, phosphate homeostasis, insulin sensitivity, immune signalling, cell cycle regulation, and ribosome synthesis. Identified more than 20 years ago, there is still only a rudimentary understanding of the mechanisms by which inositol pyrophosphates participate in these myriad pathways governing cell physiology and homeostasis. The unique stereochemical and bioenergetic properties these molecules possess as a consequence of the presence of one or two pyrophosphate moieties in the vicinity of densely packed monophosphates are likely to form the molecular basis for their participation in multiple signalling and metabolic pathways. The aim of this review is to provide first time researchers in this area with an introduction to inositol pyrophosphates and a comprehensive overview on their cellular functions.

Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae

Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport

Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150Glued subunit of dynactin. IC–p150Glued interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport.

Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migration and invasion, conferring protection from aerodigestive tract carcinoma in mice

Inositol hexakisphosphate kinases (IP6Ks), a family of enzymes found in all eukaryotes, are responsible for the synthesis of 5-diphosphoinositol pentakisphosphate (5-IP7) from inositol hexakisphosphate (IP6). Three isoforms of IP6Ks are found in mammals, and gene deletions of each isoform lead to diverse, non-overlapping phenotypes in mice. Previous studies show a facilitatory role for IP6K2 in cell migration and invasion, properties that are essential for the early stages of tumorigenesis. However, IP6K2 also has an essential role in cancer cell apoptosis, and mice lacking this protein are more susceptible to the development of aerodigestive tract carcinoma upon treatment with the oral carcinogen 4-nitroquinoline-1-oxide (4NQO). Not much is known about the functions of the equally abundant and ubiquitously expressed IP6K1 isoform in cell migration, invasion and cancer progression. We conducted a gene expression analysis on mouse embryonic fibroblasts (MEFs) lacking IP6K1, revealing a role for this protein in cell receptor-extracellular matrix interactions that regulate actin cytoskeleton dynamics. Consequently, cells lacking IP6K1 manifest defects in adhesion-dependent signaling, evident by lower FAK and Paxillin activation, leading to reduced cell spreading and migration. Expression of active, but not inactive IP6K1 reverses migration defects in IP6K1 knockout MEFs, suggesting that 5-IP7 synthesis by IP6K1 promotes cell locomotion. Actin cytoskeleton remodeling and cell migration support the ability of cancer cells to achieve their complete oncogenic potential. Cancer cells with lower IP6K1 levels display reduced migration, invasion, and anchorage-independent growth. When fed an oral carcinogen, mice lacking IP6K1 show reduced progression from epithelial dysplasia to invasive carcinoma. Thus, our data reveal that like IP6K2, IP6K1 is also involved in early cytoskeleton remodeling events during cancer progression. However, unlike IP6K2, IP6K1 is essential for 4NQO-induced invasive carcinoma. Our study therefore uncovers similarities and differences in the roles of IP6K1 and IP6K2 in cancer progression, and we propose that an isoform-specific IP6K1 inhibitor may provide a novel route to suppress carcinogenesis.

IP6K1 is essential for chromatoid body formation and temporal regulation of Tnp2 and Prm2 expression in mouse spermatids

ABSTRACT Inositol hexakisphosphate kinases (IP6Ks) are enzymes that synthesise the inositol pyrophosphate 5-diphosphoinositol pentakisphosphate (5-IP7), which is known to regulate several physiological processes. Deletion of IP6K1, but not other IP6K isoforms, causes sterility in male mice. Here, we present a detailed investigation of the specific function of IP6K1 in spermatogenesis. Within the mouse testis, IP6K1 is expressed at high levels in late stage pachytene spermatocytes and in round spermatids. We found IP6K1 to be a novel component of the chromatoid body, a cytoplasmic granule found in round spermatids that is composed of RNA and RNA-binding proteins, and noted that this structure is absent in Ip6k1−/− round spermatids. Furthermore, juvenile spermatids from Ip6k1−/− mice display premature expression of the transition protein TNP2 and the protamine PRM2 due to translational derepression. The aberrant localisation of these key sperm-specific chromatin components, together with the persistence of somatic histones, results in abnormal spermatid elongation, failure to complete spermatid differentiation and azoospermia in these mice. Our study thus identifies IP6K1 as an indispensable factor in the temporal regulation of male germ cell differentiation. This article has an associated First Person interview with the first author of the paper. Summary: Deletion of inositol hexakisphosphate kinase 1 (IP6K1) results in the absence of chromatoid bodies and premature translation of Tnp2 and Prm2, leading to arrested differentiation in mouse spermatids.

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules

AbstractInositol pyrophosphates (PP-IPs) are a class of energy-rich signalling molecules found in all eukaryotic cells. These are derivatives of inositol that contain one or more diphosphate (or pyrophosphate) groups in addition to monophosphates. The more abundant and best studied PP-IPs are diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). These molecules can influence protein function by two mechanisms: binding and pyrophosphorylation. The former involves the specific interaction of a particular inositol pyrophosphate with a binding site on a protein, while the latter is a unique attribute of inositol pyrophosphates, wherein the β-phosphate moiety is transferred from a PP-IP to a pre-phosphorylated serine residue in a protein to generate pyrophosphoserine. Both these events can result in changes in the target protein’s activity, localisation or its interaction with other partners. As a consequence of their ubiquitous presence in all eukaryotic organisms and all cell types examined till date, and their ability to modify protein function, PP-IPs have been found to participate in a wide range of metabolic, developmental, and signalling pathways. This review highlights many of the known functions of PP-IPs in the context of their temporal and spatial distribution in eukaryotic cells.

Deacetylation of catalytic lysine in CDK1 is essential for Cyclin-B binding and cell cycle

Cyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.