Insights into the roles of inositol hexakisphosphate kinase 1 (IP6K1) in mammalian cellular processes.

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation.

Engineering the thermal stability of a polyphosphate kinase by ancestral sequence reconstruction to expand the temperature boundary for an industrially applicable ATP regeneration system.

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effectively avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications.IMPORTANCEATP regeneration is an important basic applied study in the field of cell-free biocatalysis. Polyphosphate kinase (PPK) is an enzyme tool widely used for energy regeneration during enzymatic reactions. However, the thermal stability of the PPKs reported to date that can efficiently regenerate ATP is usually poor, which greatly limits their application. In this study, the thermal stability of a difficult-to-engineer PPK from Sulfurovum lithotrophicum was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides.

A novel homozygous variant in PMVK is associated with enhanced IL1ß secretion and a hyper-IgD syndrome-like phenotype.

The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate-limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper-IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS-like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1ß, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants.

Genes controlling hydrolysate toxin tolerance identified by QTL analysis of the natural Saccharomyces cerevisiae BCC39850.

Lignocellulosic material can be converted to valorized products such as fuels. Pretreatment is an essential step in conversion, which is needed to increase the digestibility of the raw material for microbial fermentation. However, pretreatment generates by-products (hydrolysate toxins) that are detrimental to microbial growth. In this study, natural Saccharomyces strains isolated from habitats in Thailand were screened for their tolerance to synthetic hydrolysate toxins (synHTs). The Saccharomyces cerevisiae natural strain BCC39850 (toxin-tolerant) was crossed with the laboratory strain CEN.PK2-1C (toxin-sensitive), and quantitative trait locus (QTL) analysis was performed on the segregants using phenotypic scores of growth (OD600) and glucose consumption. VMS1, DET1, KCS1, MRH1, YOS9, SYO1, and YDR042C were identified from QTLs as candidate genes associated with the tolerance trait. CEN.PK2-1C knockouts of the VMS1, YOS9, KCS1, and MRH1 genes exhibited significantly greater hydrolysate toxin sensitivity to growth, whereas CEN.PK2-1C knock-ins with replacement of VMS1 and MRH1 genes from the BCC39850 alleles showed significant increased ethanol production titers compared with the CEN.PK2-1C parental strain in the presence of synHTs. The discovery of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin tolerance in S. cerevisiae indicates the roles of the endoplasmic-reticulum-associated protein degradation pathway, plasma membrane protein association, and the phosphatidylinositol signaling system in this trait. KEY POINTS: • QTL analysis was conducted using a hydrolysate toxin-tolerant S. cerevisiae natural strain • Deletion of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin-sensitivity • Replacement of VMS1 and MRH1 with natural strain alleles increased ethanol production titers in the presence of hydrolysate toxins.

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

A Novel PMVK Variant Associated with Familial Porokeratosis.

BACKGROUND: Porokeratosis is a rare chronic progressive hypokeratotic skin disease, possibly related to the mevalonate pathway. Variations in four enzymes, including phosphomevalonate kinase (PMVK) may alter this pathway, ultimately leading to porokeratosis. OBJECTIVES: The aim of the study was to identify the causative gene variant of porokeratosis in a Chinese family and investigate its population frequency and pathogenicity. METHOD: In this study, Sanger sequencing was used to identify the gene variant causative of porokeratosis; its population frequency was investigated by polymerase chain reaction-restriction fragment length polymorphism in 4 patients and three normal individuals as well as in 100 normal unrelated controls; finally, the pathogenicity of the mutation and the associated structural changes were predicted. RESULTS: We identified a novel heterozygous missense variant, c.207G>T (p. Lys69Asn) in the PMVK gene. This variant was found in all patients but not in the normal individuals in this family or in the 100 controls. In silico analysis indicated that the variant was pathogenic; p.Lys69Asn changed the length of the α-helix and the hydrogen bond pattern compared with the wild-type protein. CONCLUSIONS: The novel variant c.207G>T (p. Lys69Asn) in the PMVK gene was the causative variant in this porokeratosis family. This finding provides further evidence for the genetic basis of this disease.

Comprehensive Analysis of Diabetes Mellitus-related Gene Expression and Associated Prognoses in Human Lung Cancer.

INTRODUCTION: Diabetes mellitus (DM) is a major public health problem worldwide. Cancer is the second most common cause of death in the United States and the leading cause of death in China. There is compelling evidence that individual risk for type 2 diabetes mellitus (T2DM) is strongly influenced by genetic factors. DM and cancer may interact with one another; some kinds of cancer accompany DM, and DM can also promote cancer. METHODS: An analysis was conducted of diabetes mellitus-related gene (DM-gene) expression levels in tumor and normal tissues, clinical parameters, tumor stages, mutations, copy number variations (CNVs), immune cell infiltration, survival, gene enrichment, and gene ontology annotations. RESULTS: This analysis revealed six genes that appear to play key roles in lung cancer survival: MTMR3 (in lung adenocarcinoma [LUAD]) and COBLL1, PPARG, PPIP5K2, RREB1, and WFS1 (in lung squamous cell carcinoma [LUSC]). CONCLUSION: The results suggested that clinical practitioners and researchers should account for PPARG and RREB1 expression when selecting or testing chemotherapy drugs.

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States.

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg2+ or Mn2+; IP6, 5-IP7, or 1,5-IP8) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP6 or 5-IP7 engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N6. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N6-benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP6, 5-IP7, and 1,5-IP8. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of pho1 Gene Expression.

Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1-385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1's kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover. IMPORTANCE Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP8 synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP6. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP8 is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP8 turnover.

Inositol hexakisphosphate kinase-2 non-catalytically regulates mitophagy by attenuating PINK1 signaling.

Inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in various cellular functions including neuroprotection. Absence of IP6K2 causes impairment of oxidative phosphorylation regulated by creatine kinase-B. In the present study, we show that IP6K2 is involved in attenuation of PINK1-mediated mitochondrial autophagy (mitophagy) in the brain. Up-regulation of dynamin-related protein (Drp-1), as well as increased expression of mitochondrial biogenesis markers (PGC1-α and NRF-1) in the cerebella of IP6K2-deleted mice (IP6K2-knockout), point to the involvement of IP6K2 in the regulation of mitochondrial fission. Knockdown of IP6K2 also leads to augmented glycolysis, potentially as a compensatory mechanism for decreased mitochondrial respiration. Overexpressing IP6K2 as well as IP6K2-kinase dead mutant in IP6K2-knockdown N2A cells reverses the expression of mitophagy markers, demonstrating that IP6K2-induced mitoprotection is catalytically/kinase independent. IP6K2 supplementation in K2-PINK1 double-knockdown N2A cells fails to reverse the expression of the mitophagic marker, LC3-II, indicating that the mitoprotective effect of IP6K2 is dependent on PINK1. Overall, our study reveals a key neuroprotective role of IP6K2 in the prevention of PINK1-mediated mitophagy in the brain.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Whole Body Ip6k1 Deletion Protects Mice from Age-Induced Weight Gain, Insulin Resistance and Metabolic Dysfunction.

(1) Background: We previously demonstrated that disruption of IP6K1 improves metabolism, protecting mice from high-fat diet-induced obesity, insulin resistance, and non-alcoholic fatty liver disease and steatohepatitis. Age-induced metabolic dysfunction is a major risk factor for metabolic diseases. The involvement of IP6K1 in this process is unknown. (2) Methods: Here, we compared body and fat mass, insulin sensitivity, energy expenditure and serum-, adipose tissue- and liver-metabolic parameters of chow-fed, aged, wild type (aWT) and whole body Ip6k1 knockout (aKO) mice. (3) Results: IP6K1 was upregulated in the adipose tissue and liver of aWT mice compared to young WT mice. Moreover, Ip6k1 deletion blocked age-induced increase in body- and fat-weight and insulin resistance in mice. aKO mice oxidized carbohydrates more efficiently. The knockouts displayed reduced levels of serum insulin, triglycerides, and non-esterified fatty acids. Ip6k1 deletion partly protected age-induced decline of the thermogenic uncoupling protein UCP1 in inguinal white adipose tissue. Targets inhibited by IP6K1 activity such as the insulin sensitivity- and energy expenditure-inducing protein kinases, protein kinase B (PKB/Akt) and AMP-activated protein kinase (AMPK), were activated in the adipose tissue and liver of aKO mice. (4) Conclusions: Ip6k1 deletion maintains healthy metabolism in aging and thus, targeting this kinase may delay the development of age-induced metabolic dysfunction.

[Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system].

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis.

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.

Pharmacological tools to investigate inositol polyphosphate kinases - Enzymes of increasing therapeutic relevance.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are a group of central eukaryotic metabolites and signaling molecules. Due to the diverse cellular functions and widespread diseases InsPs and PP-InsPs are associated with, pharmacological targeting of the kinases involved in their biosynthesis has become a significant research interest in the last decade. In particular, the development of inhibitors for inositol hexakisphosphate kinases (IP6Ks) has leaped forward, while other inositol phosphate kinases have received scant attention. This review summarizes the efforts undertaken so far for discovering potent and selective inhibitors for this diverse group of small molecule kinases. The benefits of pharmacological inhibition are highlighted, given the multiple kinase-independent functions of inositol phosphate kinases. The distinct structural families of InsP and PP-InsP kinases are presented, and we discuss how compound availability for different inositol phosphate kinase families varies drastically. Lead compound discovery and optimization for the inositol kinases would benefit from detailed structural information on the ATP-binding sites of these kinases, as well as reliable biochemical and cellular read-outs to monitor inositol phosphate kinase activity in complex settings. Efforts to further tune well-established inhibitors, while simultaneously reviving tool compound development for the more neglected kinases from this family are indisputably worthwhile, considering the large potential therapeutic benefits.

Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis.

OBJECTIVE: Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH. METHODS: Body weight and composition; energy expenditure; glycemic profiles; and serum and liver metabolic, inflammatory, fibrotic and toxicity parameters were assessed in mice fed Western and high-fructose diet (HFrD) (WD: 40% kcal fat, 1.25% cholesterol, no added choline and HFrD: 60% kcal fructose). Mitochondrial oxidative capacity was evaluated in isolated hepatocytes. RNA-Seq was performed in liver samples. Livers from human NASH patients were analyzed by immunoblotting and mass spectrometry. RESULTS: HKO mice displayed increased hepatocyte mitochondrial oxidative capacity and improved insulin sensitivity but were not resistant to body weight gain. Improved hepatocyte metabolism partially protected HKO mice from NAFLD/NASH. In contrast, enhanced whole-body metabolism and reduced body fat accumulation significantly protected whole-body Ip6k1-KO mice from NAFLD/NASH. Mitochondrial oxidative pathways were upregulated, whereas gluconeogenic and fibrogenic pathways were downregulated in Ip6k1-KO livers. Furthermore, IP6K1 was upregulated in human NASH livers and interacted with the enzyme O-GlcNAcase that reduces protein O-GlcNAcylation. Protein O-GlcNAcylation was found to be reduced in Ip6k1-KO and HKO mouse livers. CONCLUSION: Pleiotropic actions of IP6K1 in the liver and other metabolic tissues mediate hepatic metabolic dysfunction and NAFLD/NASH, and thus IP6K1 deletion may be a potential treatment target for this disease.

PPIP5K2 promotes colorectal carcinoma pathogenesis through facilitating DNA homologous recombination repair.

Colorectal carcinoma (CRC) is the second most deadly cancer worldwide. Therapies that take advantage of DNA repair defects have been explored in various tumors but not yet systematically in CRC. Here, we found that Diphosphoinositol Pentakisphosphate Kinase 2 (PPIP5K2), an inositol pyrophosphate kinase, was highly expressed in CRC and associated with a poor prognosis of CRC patients. In vitro and in vivo functional studies demonstrated that PPIP5K2 could promote the proliferation and migration ability of CRC cells independent of its inositol pyrophosphate kinase activity. Mechanically, S1006 dephosphorylation of PPIP5K2 could accelerate its dissociation with 14-3-3 in the cytoplasm, resulting in more nuclear distribution. Moreover, DNA damage treatments such as doxorubicin (DOX) or irradiation (IR) could induce nuclear translocation of PPIP5K2, which subsequently promoted homologous recombination (HR) repair by binding and recruiting RPA70 to the DNA damage site as a novel scaffold protein. Importantly, we verified that S1006 dephosphorylation of PPIP5K2 could significantly enhance the DNA repair ability of CRC cells through a series of DNA repair phenotype assays. In conclusion, PPIP5K2 is critical for enhancing the survival of CRC cells via facilitating DNA HR repair. Our findings revealed an unrecognized biological function and mechanism model of PPIP5K2 dependent on S1006 phosphorylation and provided a potential therapeutic target for CRC patients.

2'-Fucosyllactose production in engineered Escherichia coli with deletion of waaF and wcaJ and overexpression of FucT2.

2'-Fucosyllactose (2'-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2'-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2'-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2'-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2'-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2'-FL production in E. coli.

Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides.

Nucleic acid derivatives are involved in cell growth and replication, but they are also particularly important as building blocks for RNA and DNA synthesis [...].