On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

Protein polyphosphorylation of lysine residues by inorganic polyphosphate.

The complexity of higher organisms is not simply a reflection of the number of genes. A network of additional regulatory features, including protein post-translational modifications (PTMs), provides functional complexity otherwise inaccessible to a single gene product. Virtually all proteins are targets of PTMs. Here we characterize "polyphosphorylation" as the covalent attachment of inorganic polyphosphate (polyP) to target proteins. We found that nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1), are polyphosphorylated. This modification occurs on lysine (K) residues within a conserved N-terminal polyacidic serine (S) and K-rich (PASK) cluster. We show that polyphosphorylation negatively regulates Nsr1/Top1 interaction and impairs Top1 enzymatic activity. Physiological modulation of cellular levels of polyP regulates Top1 activity by modifying its polyphosphorylation status. We propose that polyphosphorylation adds an additional layer of regulation to nuclear signaling, where many PASK-containing proteins are known to play important roles.

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.

Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation.

A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.

Genome editing for resistance against plant pests and pathogens.

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles.

Sorting endosomes (SEs) are the regulatory hubs for sorting cargo to multiple organelles, including lysosome-related organelles, such as melanosomes in melanocytes. In parallel, melanosome biogenesis is initiated from SEs with the processing and sequential transport of melanocyte-specific proteins toward maturing melanosomes. However, the mechanism of cargo segregation on SEs is largely unknown. Here, RNAi screening in melanocytes revealed that knockdown of Rab4A results in defective melanosome maturation. Rab4A-depletion increases the number of vacuolar endosomes and disturbs the cargo sorting, which in turn lead to the mislocalization of melanosomal proteins to lysosomes, cell surface and exosomes. Rab4A localizes to the SEs and forms an endosomal complex with the adaptor AP-3, the effector rabenosyn-5 and the motor KIF3, which possibly coordinates cargo segregation on SEs. Consistent with this, inactivation of rabenosyn-5, KIF3A or KIF3B phenocopied the defects observed in Rab4A-knockdown melanocytes. Further, rabenosyn-5 was found to associate with rabaptin-5 or Rabip4/4' (isoforms encoded by Rufy1) and differentially regulate cargo sorting from SEs. Thus, Rab4A acts a key regulator of cargo segregation on SEs.This article has an associated First Person interview with the first author of the paper.

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.

Copper Complexes with 1,10-Phenanthroline Derivatives: Underlying Factors Affecting Their Cytotoxicity.

The interpretation of in vitro cytotoxicity data of Cu(II)-1,10-phenanthroline (phen) complexes normally does not take into account the speciation that complexes undergo in cell incubation media and its implications in cellular uptake and mechanisms of action. We synthesize and test the activity of several distinct Cu(II)-phen compounds; up to 24 h of incubation, the cytotoxic activity differs for the Cu complexes and the corresponding free ligands, but for longer incubation times (e.g., 72 h), all compounds display similar activity. Combining the use of several spectroscopic, spectrometric, and electrochemical techniques, the speciation of Cu-phen compounds in cell incubation media is evaluated, indicating that the originally added complex almost totally decomposed and that Cu(II) and phen are mainly bound to bovine serum albumin. Several methods are used to disclose relationships between structure, activity, speciation in incubation media, cellular uptake, distribution of Cu in cells, and cytotoxicity. Contrary to what is reported in most studies, we conclude that interaction with cell components and cell death involves the separate action of Cu ions and phen molecules, not [Cu(phen)n] species. This conclusion should similarly apply to many other Cu-ligand systems reported to date.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis.

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

The new world of inorganic polyphosphates.

Post-translational modifications (PTMs) add regulatory features to proteins that help establish the complex functional networks that make up higher organisms. Advances in analytical detection methods have led to the identification of more than 200 types of PTMs. However, some modifications are unstable under the present detection methods, anticipating the existence of further modifications and a much more complex map of PTMs. An example is the recently discovered protein modification polyphosphorylation. Polyphosphorylation is mediated by inorganic polyphosphate (polyP) and represents the covalent attachment of this linear polymer of orthophosphate to lysine residues in target proteins. This modification has eluded MS analysis as both polyP itself and the phosphoramidate bonds created upon its reaction with lysine residues are highly unstable in acidic conditions. Polyphosphorylation detection was only possible through extensive biochemical characterization. Two targets have been identified: nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1). Polyphosphorylation occurs within a conserved N-terminal polyacidic serine (S) and lysine (K) rich (PASK) cluster. It negatively regulates Nsr1-Top1 interaction and impairs Top1 enzymatic activity, namely relaxing supercoiled DNA. Modulation of cellular levels of polyP regulates Top1 activity by modifying its polyphosphorylation status. Here we discuss the significance of the recently identified new role of inorganic polyP.

Phosphate, inositol and polyphosphates.

Eukaryotic cells have ubiquitously utilized the myo-inositol backbone to generate a diverse array of signalling molecules. This is achieved by arranging phosphate groups around the six-carbon inositol ring. There is virtually no biological process that does not take advantage of the uniquely variable architecture of phosphorylated inositol. In inositol biology, phosphates are able to form three distinct covalent bonds: phosphoester, phosphodiester and phosphoanhydride bonds, with each providing different properties. The phosphoester bond links phosphate groups to the inositol ring, the variable arrangement of which forms the basis of the signalling capacity of the inositol phosphates. Phosphate groups can also form the structural bridge between myo-inositol and diacylglycerol through the phosphodiester bond. The resulting lipid-bound inositol phosphates, or phosphoinositides, further expand the signalling potential of this family of molecules. Finally, inositol is also notable for its ability to host more phosphates than it has carbons. These unusual organic molecules are commonly referred to as the inositol pyrophosphates (PP-IPs), due to the presence of high-energy phosphoanhydride bonds (pyro- or diphospho-). PP-IPs themselves constitute a varied family of molecules with one or more pyrophosphate moiety/ies located around the inositol. Considering the relationship between phosphate and inositol, it is no surprise that members of the inositol phosphate family also regulate cellular phosphate homoeostasis. Notably, the PP-IPs play a fundamental role in controlling the metabolism of the ancient polymeric form of phosphate, inorganic polyphosphate (polyP). Here we explore the intimate links between phosphate, inositol phosphates and polyP, speculating on the evolution of these relationships.

Inositol pyrophosphates regulate JMJD2C-dependent histone demethylation.

Epigenetic modifications of chromatin represent a fundamental mechanism by which eukaryotic cells adapt their transcriptional response to developmental and environmental cues. Although an increasing number of molecules have been linked to epigenetic changes, the intracellular pathways that lead to their activation/repression have just begun to be characterized. Here, we demonstrate that inositol hexakisphosphate kinase 1 (IP6K1), the enzyme responsible for the synthesis of the high-energy inositol pyrophosphates (IP7), is associated with chromatin and interacts with Jumonji domain containing 2C (JMJD2C), a recently identified histone lysine demethylase. Reducing IP6K1 levels by RNAi or using mouse embryonic fibroblasts derived from ip6k1(-/-) knockout mice results in a decreased IP7 concentration that epigenetically translates to reduced levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increased levels of acetyl-H3K9. Conversely, expression of IP6K1 induces JMJD2C dissociation from chromatin and increases H3K9me3 levels, which depend on IP6K1 catalytic activity. Importantly, these effects lead to changes in JMJD2C-target gene transcription. Our findings demonstrate that inositol pyrophosphate signaling influences nuclear functions by regulating histone modifications.

Functions of inorganic polyphosphates in eukaryotic cells: a coat of many colours.

PolyP (inorganic polyphosphate) is a linear polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. This polymer is present in all living organisms from bacteria to mammals. Until recently, most of the studies on polyP have focused on its function in prokaryotes. In prokaryotes, polyP has been implicated in many unrelated processes ranging from basic metabolism to structural functions. However, polyP analysis and function in higher eukaryotes has been gaining momentum recently. In the present review, we mainly aim to discuss the proposed intracellular functions of polyP in higher eukaryotes and its detection methods.

A mammalian model reveals inorganic polyphosphate channeling into the nucleolus and induction of a hyper-condensate state.

Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus.

Tumors of the foot and ankle: a single-institution experience.

Tumors of the foot and ankle are rare, and the particular clinicopathologic features, therapeutic approach, and outcomes in this setting are not well established. From January 2000 to December 2010, 72 patients with primary musculoskeletal tumors of the foot and ankle, both benign and malignant, were treated at a single institution. Of the 72 patients, 56% were female. The median age was 52 years. Of the 72 tumors, 62 (86.11%) were located in the foot and 10 were located in the ankle; 63 (87.5%) were soft tissue tumors and 9 (12.5%) were bone tumors. Overall, 56 (78%) were benign tumors and 16 (22%) were malignant tumors. The most frequent soft tissue and bone diagnosis was giant cell tumor. The median follow-up period was 49 months. The vast majority of the tumors were located in the foot. Benign tumors were dominant, outnumbering malignant tumors by more than 3 to 1. The diversity of the histologic benign types was evident, with giant cell tumor, angiomyoma, and lipoma the most frequent. Regarding the malignant tumors, a clear male predominance was present, the median age was 45 years, and the most frequent tumor was synoviosarcoma. The 9-year overall and disease-free survival rate was 65% and 40%, respectively.

Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases.

Inorganic polyphosphate (poly-P) consists of just a chain of phosphate groups linked by high energy bonds. It is found in every organism and is implicated in a wide variety of cellular processes (e.g. phosphate storage, blood coagulation, and pathogenicity). Its metabolism has been studied mainly in bacteria while remaining largely uncharacterized in eukaryotes. It has recently been suggested that poly-P metabolism is connected to that of highly phosphorylated inositol species (inositol pyrophosphates). Inositol pyrophosphates are molecules in which phosphate groups outnumber carbon atoms. Like poly-P they contain high energy bonds and play important roles in cell signaling. Here, we show that budding yeast mutants unable to produce inositol pyrophosphates have undetectable levels of poly-P. Our results suggest a prominent metabolic parallel between these two highly phosphorylated molecules. More importantly, we demonstrate that DDP1, encoding diadenosine and diphosphoinositol phosphohydrolase, possesses a robust poly-P endopolyphosphohydrolase activity. In addition, we prove that this is an evolutionarily conserved feature because mammalian Nudix hydrolase family members, the three Ddp1 homologues in human cells (DIPP1, DIPP2, and DIPP3), are also capable of degrading poly-P.

Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.

High-energy inositol pyrophosphates, such as IP(7) (diphosphoinositol pentakisphosphate), can directly donate a beta-phosphate to a prephosphorylated serine residue generating pyrophosphorylated proteins. Here, we show that the beta subunit of AP-3, a clathrin-associated protein complex required for HIV-1 release, is a target of IP(7)-mediated pyrophosphorylation. We have identified Kif3A, a motor protein of the kinesin superfamily, as an AP3B1-binding partner and demonstrate that Kif3A, like the AP-3 complex, is involved in an intracellular process required for HIV-1 Gag release. Importantly, IP(7)-mediated pyrophosphorylation of AP3B1 modulates the interaction with Kif3A and, as a consequence, affects the release of HIV-1 virus-like particles. This study identifies a cellular process that is regulated by IP(7)-mediated pyrophosphorylation.

The developmental quality of participation experiences: beyond the rhetoric that "participation is always good!".

A powerful rhetoric regarding the importance of adolescents' civic engagement and political participation is common in contemporary societies, whilst citizens, both adolescent and adults, seem to express a growing scepticism and alienation regarding politics. Even if this disengagement is debatable, as there are simultaneous signs of an increasing involvement in a variety of emerging and broadly-defined civic and political activities, we argue that the benefits of these experiences should be scrutinized using psychological evidence-based criteria. We rest on classical contributions from developmental psychology, educational theory and political science to define criteria that could inform the quality of participation experiences, and then present two studies that explore its adequacy. Study 1 is a cross-sectional study that observes that higher quality civic and political experiences are connected with more complex modes of thinking about politics. In Study 2, a two-wave longitudinal design, the quality of participation experiences is a significant predictor of change patterns of political attitudes; moreover, results support the argument that participation is not good in itself and that some experiences, with lesser developmental quality, might have a detrimental effect on adolescents' political development.

Exercise training enhances autonomic function after acute myocardial infarction: a randomized controlled study.

INTRODUCTION: Heart rate recovery, defined as the fall in heart rate during the first minute after exercise, is an indicator of autonomic function, and has been found to be an independent predictor of mortality after acute myocardial infarction. Exercise training has several well-known benefits in terms of cardiorespiratory fitness, modifiable cardiovascular risk factors and prognosis after acute coronary events. However, there are no randomized controlled studies in the literature evaluating the effects of exercise training per se, controlling for changes in medication and diet, on heart rate recovery. Thus, this study aims to assess the effects of exercise training on autonomic function in coronary artery disease patients recovering from acute myocardial infarction. METHODS: Thirty-eight patients following a first acute myocardial infarction participated in this prospective randomized clinical trial. Patients were randomized into two groups: exercise training or control. The exercise group participated in an 8-week aerobic exercise program, while the control received standard medical care and follow-up. Changes in hemodynamics at rest and at peak exercise (heart rate, systolic and diastolic blood pressure, and rate pressure product), dietary intake, cardiorespiratory fitness, and heart rate recovery were assessed. RESULTS: Medication and diet remained unchanged in both groups during the study period. The exercise-training group improved resting hemodynamics, particularly resting heart rate (from 68.0 ± 9.2 to 62.6 ± 8.7 bpm, p=0.030) and systolic blood pressure (from 135 ± 7.1 to 125.6 ± 11.3 mm Hg, p=0.012), cardiorespiratory fitness (from 30.8 ± 7.8 to 33.9 ± 8.3 ml/min/kg, p=0.016), and heart rate recovery (from 20 ± 6 to 24 ± 5 bpm, p=0.007). No significant changes were observed in the control group. CONCLUSIONS: Exercise training improved autonomic function, assessed by heart rate recovery, resting heart rate and systolic blood pressure, in the absence of changes in diet or medication.