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.

The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions.

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH3) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

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.

Pandemia: vivências de médicos da atenção primária à saúde e de mestrandos em Saúde da Família / Pandemic: experiences of primary health care doctors and master's degree students in family health

Resumo: Introdução: A Covid-19 trouxe uma série de desafios para o Sistema Único de Saúde. Na atenção primária à saúde (APS), tais desafios se somam aos já existentes. Objetivo: Este estudo teve como objetivo analisar a vivência de enfrentamento da pandemia de Covid-19 entre médicos do Programa Mais Médicos Brasil, mestrandos em Saúde da Família e atuantes na APS. Método: Trata-se de estudo qualitativo envolvendo oito médicos da APS de Alagoas que cursam o Mestrado Profissional em Saúde da Família (PROFSAÚDE). Foram elaboradas cinco questões, cujas respostas foram analisadas com base na teoria de Análise de Conteúdo. Resultado: Observaram-se três categorias e quatro subcategorias: categoria 1 - "Processo de estudo e de trabalho" (subcategoria 1.1 - "Características do PROFSAÚDE" - e subcategoria 1.2 - "Estratégias pessoais desenvolvidas"), categoria 2 - "Desafios no gerenciamento da vida" (subcategoria 2.1 - "Mudanças no cotidiano" - e subcategoria 2.2 - "Impacto nas emoções") e categoria 3 - "Crescimento pessoal e profissional". Conclusão: Em tempos de pandemia, os profissionais médicos vivenciam situações complexas e dinâmicas em razão de um duplo e acumulativo processo - o trabalho na APS e a condição de mestrando. Apesar de todas as dificuldades enfrentadas, o mestrado possibilitou o aprimoramento das habilidades em lidar com situações críticas.

Abstract: Introduction: COVID-19 has brought numerous challenges for the Health System in Brazil. In Primary Health Care, these challenges add to those that already exist. Objective: To analyze the experience of facing the COVID-19 pandemic among doctors of the Mais Médicos Brasil Program and master's degree students in Family Health and those working in Primary Health Care. Methods: Qualitative study involving eight doctors from Primary Health Care in Alagoas who are also studying the professional master's degree in Family Health (PROFSAÚDE). Five questions were developed, the answers to which were analyzed based on Content Analysis theory. Results: Three categories and four subcategories were observed: Category 1 - Study and work process (Subcategory 1.1- Characteristics of PROFSAÚDE; Subcategory 1.2 - Personal strategies developed); Category 2 - Challenges in Life Management (Subcategory 2.1 - Changes in daily life; Subcategory 2.2 - Impact on emotions) and Category 3 - Personal and Professional Growth. Conclusion: During the pandemic, medical professionals experience complex and dynamic situations due to a dual and cumulative process - working in PHC and studying for their master's degree. Despite all the difficulties faced, the master's degree allowed them to improve skills in dealing with critical situations.

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.

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.

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.

Concepções sobre o processo de alta hospitalar: uma revisão crítica / Conceptions on patient discharge process: a critical review / Concepciones alta del paciente: una revisión crítica

Objetivamos discutir as concepções acerca do processo de alta hospitalar, a partir de uma revisão crítica da literatura científica em diálogocom a experiência vivenciada pelas autoras, no Programa de Residência Multiprofissional em Saúde do Adulto e do Idoso de um Estado da região Nordeste do Brasil. A busca das produções científicas foi realizada em bases de dados (Scielo; Bireme e PubMed), utilizando os descritores alta hospitalar e alta do paciente, separadamente. Foram encontrados um total de cinquenta e quatro produções, no entanto, vinte artigos nortearam a proposta deste trabalho. Para análise do material, fizemos uma leitura seletiva a qual categorizou e relacionou as produções norteada ao objetivo.Notamos predominância de pesquisas nas áreas de Enfermagem e Medicina, que estão publicadas em revistas das áreas correspondentes, durante o período de 2010 a 2013. Esses estudos, em sua maioria,caracterizam-se como epidemiológicos e descritivos, com utilização de escalas eforam realizados predominantementeno Estado de São Paulo. Identificamos que as mudanças na concepção de alta hospitalar produzem efeitos na maneira de desenvolver as práticas. Ao refletir sobre a literatura científica e nossas práticas, compreendemos a alta como um processo e não apenas, como a saída do paciente do hospital. Por fim, demarcamos que operar com um ou outro conceito, não é apenas uma questão teórica, e sim, o reflexo do modo como nossos saberes produzem formas de intervenção na realidade e de relação com os sujeitos. (AU)

We aim to discuss the conceptions about the hospital discharge process, based on a critical review of the scientific literature in dialogue with the experience lived by the authors, in the Program of Multiprofessional Residency in Adult Health and the Elderly of a State in the Northeast region of Brazil. The research for the scientific productions was carried out in databases (Scielo; Bireme and PubMed), using the descriptors discharge hospital and discharge of the patient, separately. A total of fifty four productions were found, however, twenty articles guided the proposal of this work. For the analysis of the material, we made a selective reading that directly related and categorized the productions to the objective. We note the predominance of researches in the areas of Nursing and Medicine, and these are also published in journals in the corresponding areas with a higher concentration of publications during the period from 2010 to 2013. The methodology used in these research consisted mostly of epidemiological and descriptive studies, with the use of scales. As study sites research was dominant in the State of São Paulo. We have identified that changes in the conception of hospital discharge produce effects in the way of developing the practices. When we reflect on the scientific literature and our practices, we understand the discharge as a process and not only, when the patient leaves the hospital. Finally, we point out that to operate with one or another concept is not only a theoretical question, but a reflection of the way our knowledge produces forms of intervention in reality and in relation to the subjects. (AU)

El objetivo del estudio es discutir las concepciones del proceso de alta del hospital a partir de una revisión crítica de la literatura científica en diálogo con la experiencia vivida por los autores, en el Programa de Residencia Multidisciplinar en Salud del Adulto y el Anciano de un estado en la región noreste de Brasil. La búsqueda de la producción científica se llevó a cabo en bases de datos (Scielo, BIREME y PubMed), utilizando los descriptores de hospital y alta del paciente por separado. Se encontró un total de cincuenta y cuatro producciones, sin embargo, veinte artículos guían el propósito de este trabajo. Para el análisis del material, hicimos una lectura selectiva de la cual clasificase las producciones y producerelacionessegún el objetivo. Observamos el predominio de la investigación en enfermería y medicina, publicadas en las revistas de las áreas correspondientes en el período de 2010 a 2013. Estos estudios caracterizan se como epidemiológicos y descriptivos y por el uso de escalas, en su mayoría desarrollados en Sao Paulo. Hemos encontrado que los cambios en la conceptualización dealta del hospital tienen efecto sobre la manera de desarrollar las prácticas. Al reflexionar sobre la literatura científica y nuestras prácticas, entendemos el alta como un proceso y no sólo como salida de un paciente del hospital. Por último, resulta que trabajar con uno u otro concepto, no es sólo una cuestión teórica, sino más bien un reflejo de cómo nuestros conocimientos producen formas de intervención en la realidad y en la relación con el sujeto. (AU)

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.

Screening a Protein Array with Synthetic Biotinylated Inorganic Polyphosphate To Define the Human PolyP-ome.

Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

Binding of vanadium to human serum transferrin - voltammetric and spectrometric studies.

Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of electrochemical techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and small-angle X-ray scattering (SAXS) data it is confirmed that both VIV and VV bind to apo-hTF and holo-hTF. The electrochemical behavior of solutions containing vanadate(V) solutions at pH=7.0, analyzed by using two different voltammetric techniques, with different time windows, at a mercury electrode, Differential Pulse Polarography (DPP) and Cyclic Voltammetry (CV), is consistent with a stepwise reduction of VV→VIV and VIV→VII. Globally the voltammetric data are consistent with the formation of 2:1 complexes in the case of the system VV-apo-hTF and both 1:1 and 2:1 complexes in the case of VV-holo-hTF; the corresponding conditional formation constants were estimated. MALDI-TOF mass spectrometric data carried out with samples of VIVOSO4 and apo-hTF and of NH4VVO3 with both apo-hTF and holo-hTF with V:hTF ratios of 3:1 are consistent with the binding of vanadium to the proteins. Additionally the SAXS data suggest that both VIVOSO4 and NaVVO3 can effectively interact with human apo-transferrin, but for holo-hTF no clear evidence was obtained supporting the existence or the absence of protein-ligand interactions. This latter data suggest that the conformation of holo-hTF does not change in the presence of either VIVOSO4 or NH4VVO3. Therefore, it is anticipated that VIV or VV bound to holo-hTF may be efficiently up-taken by the cells through receptor-mediated endocytosis of hTF.

Microbial inositol polyphosphate metabolic pathway as drug development target.

Inositol polyphosphates are a diverse and multifaceted class of intracellular messengers omnipresent in eukaryotic cells. These water-soluble molecules regulate many aspects of fundamental cell physiology. Removing this metabolic pathway is deleterious: inositol phosphate kinase null mutations can result in lethality or substantial growth phenotypes. Inositol polyphosphate synthesis occurs through the actions of a set of kinases that phosphorylate phospholipase-generated IP3 to higher phosphorylated forms, such as the fully phosphorylated IP6 and the inositol pyrophosphates IP7 and IP8. Unicellular organisms have a reduced array of the kinases for synthesis of higher phosphorylated inositol polyphosphates, while human cells possess two metabolic routes to IP6. The enzymes responsible for inositol polyphosphate synthesis have been identified in all eukaryote genomes, although their amino acid sequence homology is often barely detectable by common search algorithms. Homology between human and microbial inositol phosphate kinases is restricted to a few catalytically important residues. Recent studies of the inositol phosphate metabolic pathways in pathogenic fungi (Cryptococcus neoformans) and protozoa (Trypanosome brucei) have revealed the importance of the highly phosphorylated inositol polyphosphates to the fitness and thus virulence of these pathogens. Given this, identification of inositol kinase inhibitors specifically targeting the kinases of pathogenic microorganisms is desirable and achievable.

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.

A Integração Ensino-Serviço e a Residência Multiprofissional em Saúde: um relato de experiência numa Unidade Básica de Saúde / The Service-Learning Integration and the Multiprofessional Residency in Health: an experience report in a Basic Health Unit / La Integración Enseñanza-Servicio y la Residencia Multiprofesional en la salud: un relato de experiencia en una Unidad Básica de Salud

Este estudo compreende um relato de experiência que tem como objetivo promover reflexões acerca da integração ensino-serviço no âmbito da Residência Multiprofissional em Saúde, no cenário de práticas de uma Unidade Básica de Saúde (UBS). Buscou-se analisar o modo como ocorreu a articulação das atividades realizadas pelos residentes com o cotidiano do serviço na UBS e descrever quais os desafios encontrados durante o período de atuação neste cenário, bem como as estratégias de enfrentamento desenvolvidas para lidar com tais dificuldades. Para realizar uma análise crítico-reflexiva das experiências vivenciadas, utilizou-se tanto a literatura científica existente sobre a temática da integração ensino-serviço, como a perspectiva do Construcionismo Social, na construção do conhecimento. A vivência da equipe de residentes corrobora com as experiências existentes na literatura científica acerca dos desafios enfrentados no contexto da integração ensino-serviço, apontando para a necessidade de uma maior aproximação entre os setores envolvidos nesse processo.

This study comprises an experience report that aims to promote reflections about the teaching-service integration in the context of the Multiprofessional Residency in Health, in the practice scenario of a Basic Health Unit (BHU). It sought to analyze the way the residents carried out the coordination of activities with the daily routine of the service at the BHU, as well as to describe the challenges encountered during the operation period in this scenario and the coping strategies developed to deal with such difficulties. In order to perform a critical and reflective analysis of the experiences, both the existing scientific literature on the subject of teaching and service integration, as the perspective of social constructionism were used. The experience of this resident team corroborates existing experiences in the scientific literature about the challenges faced in the context of teaching-service integration, pointing to the need for a closer relationship among the sectors involved in this process.

Este estudio comprende un relato de experiencia cuyo objetivo es promover reflexiones sobre la integración enseñanza-servicio dentro de Residencia Multiprofesional en Salud en el escenario de prácticas de una Unidad Básica de Salud (UBS). Se trató de analizar cómo fue la articulación de las actividades llevadas a cabo por los residentes con la rutina de servicio de UBS, y para describir lo que los retos encontrados durante el período de operación en este escenario y las estrategias de supervivencia desarrolladas para hacer frente a estas dificultades. Para realizar un análisis crítico y reflexivo de las experiencias pasadas, se utilizó tanto en la literatura científica existente sobre el tema de la integración enseñanza y el servicio, ya que el punto de vista del Construccionismo Social. La experiencia de este equipo residente corrobora las experiencias existentes en la literatura científica acerca de los desafíos que enfrentan en el contexto de la integración enseñanza-servicio, que apunta a la necesidad de estrechar los vínculos entre los sectores involucrados en este proceso.

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.

Why always lysine? The ongoing tale of one of the most modified amino acids.

The complex physiology of living organisms must be finely-tuned to permit the flexibility required to respond to the changing environment. Evolution has provided an interconnected and intricate array of regulatory mechanisms to facilitate this fine-tuning. The number of genes cannot alone explain the complexity of these mechanisms. Rather, signalling is regulated at multiple levels, from genomic to transcriptional, translational and post-translational. Post-translational modification (PTM) of proteins offers an additional level of regulation after protein synthesis that allows a rapid, controlled and reversible response to environmental cues. Many amino acid side chains are post-translationally modified. These modifications can either be enzymatic, such as the phosphorylation of serine, threonine and tyrosine residues, or non-enzymatic, such as the nitrosylation of cysteine residues. Strikingly, lysine residues are targeted by a particularly high number of PTMs including acetylation, methylation, ubiquitination and sumoylation. Additionally, lysines have recently been identified as the target of the non-enzymatic PTM polyphosphorylation. This novel PTM sees linear chains of inorganic polyphosphates (polyP) covalently attached to lysine residues. Interestingly, polyphosphorylation is indirectly dependent on inositol pyrophosphates, a class of cellular messengers. The attachment of polyP to lysine occurs through the phosphoramidate bond, which, unlike the phosphester bond, is unstable under the conditions used in common mass spectroscopy. This characteristic, together with the diversity of lysine PTMs, suggests that many other lysine modifications may still remain unidentified, raising the intriguing possibility that lysine PTMs may be the major means by which signalling pathways modify protein behaviour.

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.

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.