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.

The Effect of Phosphorus Metabolism on the Motion of Saccharomyces cerevisiae Volutin Granules

It is known that moving volutin granules (“dancing bodies”), mechanism of which occurrence remains poorly understood, can be observed in yeast vacuoles. This study was performed to reveal the presence of a connection between moving volutin granules of Saccharomyces cerevisiae and polyphosphate metabolism in conditions of phosphoric starvation and hypercompensation. Methods. Cytological, biochemical, statistical methods were used in the study. Results. It was observed that the inactivation of the PPN1 gene, which encodes exopolyphosphatase Ppn1, resulted in a change in the number of cells with moving volutin granules (“dancing bodies” index) in the studied conditions. The index of “dancing bodies” was almost always lower in mutant CRN strain than in parent CRY strain. Using linear correlation analysis and factor analysis with the method of principal component, it was established that the “dancing bodies” index in both strains had significant correlation coefficients with exopolyphosphatase activity (EPPA) and the content of polyphosphate fractions (polyP). The difference was that this index in parent strain correlated better with the first three fractions of inorganic polyphosphates, while in mutant strain – with polyP4 and EPPA. Conclusions. Obtained data indicated the direct connection of motion of volutin granules with phosphoric metabolism in the studied conditions. It is assumed that the phenomenon of “dancing bodies” may be a consequence of the activity of vacuolar polyphosphatases.

Dynamic polyphosphate metabolism coordinating with manganese ions defends against oxidative stress in the extreme bacterium Deinococcus radiodurans

Deinococcus radiodurans is an extreme bacterium with unparalleled resistance to oxidative stresses. Accumulation of intracellular Mn2+ complexing with small metabolites is the key contributor to the tolerance of D. radiodurans against oxidative stress. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn-complex in D. radiodurans remain unclear. We identified an evolutionarily ancient and negatively charged phosphate polymer (polyphosphate, PolyP) in D. radiodurans. We investigated the PolyP metabolism in the response of D. radiodurans to oxidative stress. The genes dr1939, encoding polyphosphatase kinase (PPKDr), and dra0185, encoding exopolyphosphatase (PPXDr), were identified. PPXDr is a novel exopolyphosphatase with a cofactor preference to Mn2+, which enhances the dimerization and activity of PPXDr to allow the effective cleavage of PolyP-Mn. PPKDr and PPXDr exhibited different dynamic expression profiles under oxidative stress. First, the ppkDr was upregulated leading to the accumulation of PolyP, which chelated large amounts of intracellular Mn ions. Subsequently, the expression level of ppkDr decreased while the ppxDr was substantially upregulated and effectively hydrolyzed inactive PolyP-Mn to release phosphate (Pi) and Mn2+, which could form into Mn-Pi complexes to scavenge O2- and protect proteins from oxidative damage. Hence, dynamic cellular PolyP metabolites complexed with free Mn ions highlight a defense strategy of D. radiodurans in response to oxidative stress. Importance Mn-phosphate complex (Mn-Pi) plays a key role in the cellular resistance of radioresistant bacteria. The evolutionarily ancient polyphosphate polymers (PolyP) could effectively chelate Mn2+ and donate phosphates. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn-Pi complex remain unclear. Here, we investigated the relationship of PolyP metabolites and Mn2+ homeostasis, and how they function to defend against oxidative stress in the radioresistant bacterium Deinococcus radiodurans. We found that PPXDr is a novel exopolyphosphatase with a cofactor preference for Mn2+, mediating PolyP-Mn degradation into Pi and Mn ions. The formed Mn-Pi complexes effectively protect proteins. The dynamic PolyP metabolism coordinating with Mn ions is a defense strategy of D. radiodurans in response to oxidative stress. The findings not only provide new insights into the resistance mechanism of the extreme bacterium D. radiodurans but also broaden our understanding of the functions of PolyP metabolism in organisms.

MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1−/− induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Xenotropic and polytropic retrovirus receptor 1 regulates procoagulant platelet polyphosphate

Polyphosphate is a procoagulant inorganic polymer of linear linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation, however the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. Here, we show that xenotropic and polytropic retrovirus receptor 1 (XPR1) regulates polyphosphate in platelets and is implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. Xpr1 mRNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism highlighting a fundamental role for phosphate homeostasis in thrombosis.

Inositol polyphosphate multikinase in adipocytes is dispensable for regulating energy metabolism and whole body metabolic homeostasis

Adipose tissue plays a central role in regulating whole body energy and glucose homeostasis at both organ and systemic levels. Inositol polyphosphates, such as 5-diphosphoinositol pentakisphosphate, reportedly control adipocyte functions and energy expenditure. However, the physiological roles of the inositol polyphosphate (IP) pathway in the adipose tissue are not yet fully defined. The aim of the present study was to test the hypothesis that inositol polyphosphate multikinase (IPMK), a key enzyme in the IP metabolism, plays a critical role in adipose tissue biology and obesity. We generated adipocyte-specific IPMK knockout ( Ipmk AKO) mice and evaluated metabolic phenotypes by measuring fat accumulation, glucose homeostasis, and insulin sensitivity in adult mice fed either a regular-chow diet or high-fat diet (HFD). Despite substantial reduction of IPMK, Ipmk AKO mice exhibited normal glucose tolerance and insulin sensitivity and did not show changes in fat accumulation in response to HFD-feeding. In addition, loss of IPMK had no major impact on thermogenic processes in response to cold exposure. Collectively, these findings suggest that adipocyte IPMK is dispensable for normal adipose tissue and its physiological functions in whole body metabolism, suggesting the complex roles that inositol polyphosphate metabolism has in the regulation of adipose tissue.

MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

ABSTRACTInositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol polyphosphate phosphatase 1 gene (MINPP1). Patients were found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We found that patient-derived and genome edited MINPP1-/- induced pluripotent stem cells (iPSCs) are not able to differentiate efficiently into neurons. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakiphosphate (IP6), detected in HEK293, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

ABSTRACT Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.