Vacuolar Proton Pump Research Articles

GENES ASSOCIATED WITH BLOOD PRESSURE TRAITS SHOW CELL TYPE-SPECIFIC EXPRESSION AND CONTROL MULTIPLE CAUSAL LINKS TO BLOOD PRESSURE REGULATION IN THE HUMAN KIDNEY

Objective: Hypertension affects 35% of the global adult population. It is a major risk factor for stroke, heart failure and coronary artery disease and is the leading preventable risk factor for premature death globally. Here we describe our approach to determine the causal pathways between genetic variants and blood pressure through the expression of kidney genes. Design and method: We used a catalogue of all known blood pressure associated genetic variants, gene expression profiles from 430 human kidney tissue samples and a single-cell gene expression data set to identify causal relationships with blood pressure using a colocalisation analysis and an ensemble of Mendelian randomisation methods. Results: By combining genome-wide genotypes and gene expression profiles from RNA-sequencing in an expression quantitative trait loci (eQTL) analysis we identify 418 genes where expression is modified by genetic variants previously associated with blood pressure traits. To investigate how these genes are expressed in the kidney we used gene expression profiles from 41,778 human renal cells and identified 390 (93%) of these genes to be expressed in these cells with 69 (18%) showing expression specific to individual renal cell types. There was a statistically significant enrichment for expression of blood pressure associated genes in juxtaglomerular cells and cells from the collecting tubule. We identified 111 genes where the eQTL and blood pressure association colocalised and then applied an ensemble of Mendelian randomisation (MR) methods to determine which genes also have a causal effect on blood pressure in the kidney. Our MR analysis identified 53 (13%) genes where expression is causally linked to blood pressure, these included: guanylate cyclase 1 (GUCY1A3), the main receptor for nitric oxide and the target of riociguat and nuclear receptor coactivator 7 (NCOA7) which regulates vacuolar proton pumping ATPase (V-ATPase) activity in the intercalated cells of the collecting duct. Conclusions: We have identified 53 kidney genes where expression is causally linked to blood pressure. This not only highlights the importance of the kidney in blood pressure regulation, but also identifies putative targets for drug development or repurposing.

Reconstitution of ATPase Activity from Individual Subunits of the Clathrin-coated Vesicle Proton Pump THE REQUIREMENT AND EFFECT OF THREE SMALL SUBUNITS

The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (VC) and a transmembranous proton channel (VB). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas VC, when separated from VB, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of VC results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the VC complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of VC, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca2+ but also in the presence of Mg2+. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of VC but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with VB and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.

A genome-wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity

Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.

Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency

Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.

Inhibitory protonové pumpy – známe je dobře? Jsou skutečně tak bezpečné? – část 1

Proton pump inhibitors (PPIs) are one of the most commonly used drug groups. More than 10% of patients being chronically treated in the adult population. Often patients with a high risk of vascular or renal impairment. In addition to the unquestionable effect in the treatment and in the prophylaxis of gastroduodenal diseases, PPIs have been promoted in combination with antithrombotic therapy in order to reduce bleeding in the gastrointestinal tract. In both of these indications, this is a chronic treatment, often for several years. Drug regulatory agencies (EMAs or FDA) warn against chronic PPIs use, warning of a number of serious side effects. However, chronic administration of PPIs is a common practice. It is therefore time to evaluate the benefit and risk of this important drug group. First of all, it should be noted that PPIs operate not only at the level of the classical gastric proton pump (H+/K+ ATPase), but also block the activity of the sister vacuolar proton pump (V-H+-ATPase) in a number of other organs or organelles, namely lysosomes of all somatic cells. Similarly, PPIs block the activity of a number of transporters and metabolic enzymes. Action at this level is likely to surprise the gastroenterologist. There is no doubt about the benefits of PPIs in the indication of treatment and prevention of ulcerative or reflux disease. However, what are the evidence to reduce the risk of gastriontestinal bleeding in antithrombotic treatment? In this area we have data at the level of observational studies, the decrease in the risk of bleeding by about a third is significant. However, with a relatively low incidence of bleeding in the gastrointestinal tract, the absolute decrease in risk is small, hovering at 0.3%. The observed number need to treat is around 250, i.e. for every 250 PPIs treated, we will prevent one bleeding (usually not critical). On the other hand, there are increasingly work that finds a higher incidence of cardiovascular events, renal failure, bronchial asthma and nervous disabilities in chronic PPIs treatment. In a population at high cardiovascular risk, i.e. in a typical population where we add antithrombotic treatment to PPIs, the risk is significant. The number need to harm value is around 50. Thus, in the chronic use of PPIs, the risk outweighs the benefit.

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Inoculation with the endophytic bacterium Herbaspirillum seropedicae promotes growth, nutrient uptake and photosynthetic efficiency in rice.

Higher vacuolar proton pump activity may increase plant energy and nutrient use efficiency and provide the nexus between plant inoculation with Herbaspirillum seropedicae and growth promotion. Global change and growing human population are exhausting arable land and resources, including water and fertilizers. We present inoculation with the endophytic plant-growth promoting bacterium (PGPB) Herbaspirillum seropedicae as a strategy for promoting growth, nutrient uptake and photosynthetic efficiency in rice (Oryza sativa L.). Because plant nutrient acquisition is coordinated with photosynthesis and the plant carbon status, we hypothesize that inoculation with H. seropedicae will stimulate proton (H+) pumps, increasing plant growth nutrient uptake and photosynthetic efficiency at low nutrient levels. Plants were inoculated and grown in pots with sterile soil for 90days. Herbaspirillum seropedicae endophytic colonization was successful and, as hypothesized, inoculation (1) stimulated root vacuolar H+ pumps (vacuolar H+-ATPase and vacuolar H+-PPase), and (2) increased plant growth, nutrient contents and photosynthetic efficiency. The results showed that inoculation with the endophytic bacterium H. seropedicae can promote plant growth, nutrient uptake and photosynthetic efficiency, which will likely result in a more efficient use of resources (nutrients and water) and higher production of nutrient-rich food at reduced economic and environmental costs.

Evaluation of drought tolerance in ZmVPP1-overexpressing transgenic inbred maize lines and their hybrids

The vacuolar proton-pumping pyrophosphatase gene (VPP) is often used to enhance plant drought tolerance via genetic engineering. In this study, the drought tolerance of four transgenic inbred maize lines overexpressing ZmVPP1 (PH4CV-T, PH6WC-T, Chang7-2-T, and Zheng58-T) and their transgenic hybrids was evaluated at various stages. Under normal and drought conditions, the height and fresh weight were greater for the four transgenic inbred maize lines than for the wild-type (WT) controls at the germination and seedling stages. Additionally, the transgenic plants exhibited enhanced photosynthetic efficiency at the seedling stage. In irrigated and non-irrigated fields, the four transgenic lines grew normally, but with increased ear weight and yield compared with the WT plants. Moreover, the ear weight and yield of the transgenic hybrids resulting from the PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W crosses increased in the non-irrigated field. Our results demonstrated that the growth and drought tolerance of four transgenic inbred maize lines with improved photosynthesis were enhanced by the overexpression of ZmVPP1. Moreover, the Chang7-2 and PH4CV transgenic lines may be useful for future genetic improvements of maize hybrids to increase drought tolerance.

Calcium-binding proteins are altered in the cerebellum in schizophrenia

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.

Shear Stress Increases V-H -ATPase and Acidic Vesicle Number Density, and p-mTORC2 Activation in Prostate Cancer Cells.

Cells in the tumor microenvironment experience mechanical stresses, such as compression generated by uncontrolled cell growth within a tissue, increased substrate stiffness due to tumor cell extracellular matrix (ECM) remodeling, and leaky angiogenic vessels which involve low fluid shear stress. With our hypothesis that shear stress increases V-H -ATPase number density in prostate cancer cells via activation of the mTORC1 and mTORC2 pathways, we demonstrated and quantified such a mechanism in prostate cancer cells. Moderately metastatic DU145 and highly metastatic PC3 prostate cancer cells were subjected to 0.05 dynes wall shear stress for 24 h, followed by immunocytochemistry and fluorescence measurements of 1 integrin, endosome, lysosome, V-H -ATPase proton pump, mTORC1, and p-mTORC2 antibodies. Post shear stress migration assays, and the effects of vacuolar proton pump inhibitor Bafilomycin A1 (60 nM, 24 h) as well as shear stress on the ICC fluorescence intensity of the proteins of interest were conducted with DU145 cells. Low fluid shear stress increases the fluorescence intensity of 1 integrin, endosome, lysosome, V-H -ATPase, mTORC1, and p-mTORC2 antibodies in PC3 and DU145 cells, and also increased cell migration. However, Bafilomycin A1 decreased fluorescence intensity of all of these proteins in DU145 cells exposed to shear stress, revealing that V-H -ATPase controls the expression of these proteins. Prostate cancer cell mechanotransduction increases endosomes, lysosomes, and proton pumps-where increases have been associated with enhanced cancer aggressiveness. We also show that the prostate cancer cell's response to force promotes the cancer drivers mTORC1 and mTORC2.

Type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruit.

The aim of this work was to evaluate the general role of the vacuolar pyrophosphatase proton pump (V-PPase) in sucrose accumulation in citrus species. First, three citrus V-PPase genes, designated CsVPP-1, CsVPP-2, and CsVPP-4, were identified in the citrus genome. CsVPP-1 and CsVPP-2 belonging to citrus type I V-PPase genes are targeted to the tonoplast, and CsVPP-4 belonging to citrus type II V-PPase genes is located in the Golgi bodies. Moreover, there was a significantly positive correlation between transcript levels of type I V-PPase genes and sucrose, rather than hexose, content in fruits of seven citrus cultivars. Drought and abscisic acid treatments significantly induced the CsVPP-1 and CsVPP-2 transcript levels, as well as the sucrose content. The overexpression of type I V-PPase genes significantly increased PPase activity, decreased pyrophosphate contents, and increased sucrose contents, whereas V-PPase inhibition produced the opposite effect in both citrus fruits and leaves. Furthermore, altering the expression levels of type I V-PPase genes significantly influenced the transcript levels of sucrose transporter genes. Taken together, this study demonstrated that CsVPP-1 and CsVPP-2 play key roles in sucrose storage in the vacuole by regulating pyrophosphate homeostasis, ultimately the sucrose biosynthesis and transcript levels of sucrose transport genes, providing a novel lead for engineering or breeding modified taste in citrus and other fruits.

Osteopetrosis in a six-month-old infant

Osteopetrosis is a severe and frequently fatal condition characterized by the formation of thick, dense bone, with a concomitant loss of bone marrow spaces secondary to defective osteoclast function or development. Clinical symptoms include pancytopenia, hepatosplenomegaly, fractures, cranial nerve impingement, and increased cranial pressure, among others. The condition requires rapid diagnosis and clinical intervention, as hematopoietic stem cell transplantation performed early in life can be curative in the majority of cases and can prevent the irreversible neurological impairment associated with the disease. We report a six-month-old male with osteopetrosis where bone marrow examination and molecular studies allowed a definitive diagnosis. Microscopic examination of a bone marrow biopsy demonstrated increased bone formation at various stages of ossification, decreased marrow spaces, and increased osteoclasts rimming the bone. Next-generation sequencing (NGS) demonstrated that the patient was homozygous for mutations in the TCIRG1 gene, which encodes an osteoclast vacuolar proton pump and is the most common mutation associated with the autosomal recessive or infantile malignant form of osteopetrosis. The patient underwent a cord blood stem cell transplant, but unfortunately expired four months after diagnosis. This case highlights the importance of bone marrow biopsy evaluation as part of the integrated approach to diagnosing osteopetrosis.

Copalyl Diphosphate Synthase Mutation Improved Salt Tolerance in Maize (Zea mays. L) via Enhancing Vacuolar Na+ Sequestration and Maintaining ROS Homeostasis.

Salinity stress impairs plant growth and causes crops to yield losses worldwide. Reduction of in vivo gibberellin acid (GA) level is known to repress plant size but is beneficial to plant salt tolerance. However, the mechanisms of in vivo GA deficiency-enhanced salt tolerance in maize are still ambiguous. In this study, we generated two independent maize knockout mutant lines of ent-copalyl diphosphate synthase (one of the key enzymes for early steps of GA biosynthesis), zmcps-1 and zmcps-7, to explore the role of GA in maize salt tolerance. The typical dwarf phenotype with lower GA content and delayed leaf senescence under salinity was observed in the mutant plants. The leaf water potential and cell turgor potential were significantly higher in zmcps-1 and zmcps-7 than in the wild type (WT) under salt stress. The mutant plants exhibited a lower superoxide anion production rate in leaves and also a downregulated relative expression level of NAPDH oxidase ZmRbohA-C than the WT maize under salt stress. Also, the mutant plants had higher enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) and higher content of soluble sugars and proline under salt stress. The Na+/K+ ratio was not significantly different between the mutant maize plants and WT plants under salt stress conditions, but the Na+ and K+ content was increased in zmcps-1 and zmcps-7 leaves and shoots. Na+ fluorescent dye staining showed that the mutant leaves have significantly higher vacuolar Na+ intensity than the WT maize. The expression level of vacuolar Na+/H+ exchanger gene ZmNHX1 and vacuolar proton pump genes ZmVP1-1 and ZmVP2 were upregulated in the zmcps-1 and zmcps-7 plants under salinity, further proving that in vivo GA deficiency enhanced vacuolar Na+ sequestration in zmcps-1 and zmcps-7 leaves cells to avoid Na+ cytotoxicity. Together, our results suggested that maintaining ROS homeostasis and enhancing vacuolar Na+ sequestration could be involved in GA deficiency-improved maize salt tolerance.

Investigating the role of a V‐ATPase in the Development of the Air Sac Primordia

Drosophila melanogaster is a commonly used organism in biological research. Their Air Sac Primordium (ASP) is analogous to the human lungs in that it supplies oxygen to the flight muscles. Furthermore, the ASP has emerged as a good structure to study cellular invasive processes with the potential to aid in our understanding of tumor metastasis. The ASP originates on the outside of the wing imaginal disc and then burrows into the wing imaginal disc as it develops. What genes are involved in this process is not well understood. A genetic screen for genes that are expressed in the ASP identified a proton pump gene encoding a V‐ATPase. The V‐ATPase is a vacuolar proton pump that plays a large array of roles from regulating the pH of cells internal space, to regulating endocytosis, to processing proteins and degradation. In insects, the V‐ATPase are important in establishing a membrane potential to drive K+ secretion. It has also been found to influence tracheal branching. Using RNA interference, the gene for the V‐ATPase was knocked down resulting in a defect in ASP development. Data from these and other experiments will be presented and their significance will be discussed.Support or Funding InformationKentucky Biomedical Research Infrastructure Network (KBRIN) GrantGatton Academy Research Internship Grant (RIG)Faculty Undergraduate Student Engagement (FUSE) Grant

Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana.

In Arabidopsis thaliana, the vacuolar proton-pumping pyrophosphatase (H+-PPase) is highly expressed in young tissues, which consume large amounts of energy in the form of nucleoside triphosphates and produce pyrophosphate (PPi) as a byproduct. We reported that excess PPi in the H+-PPase loss-of-function fugu5 mutant severely compromised gluconeogenesis from seed storage lipids, arrested cell division in cotyledonary palisade tissue, and triggered compensated cell enlargement; this phenotype was recovered upon sucrose supply. Thus, we provided evidence that the hydrolysis of inhibitory PPi, rather than vacuolar acidification, is the major contribution of H+-PPase during seedling establishment. Here, examination of the epidermis revealed that fugu5 pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from fugu5 background by the yeast cytosolic PPase IPP1, in fugu5-1 AVP1pro::IPP1 transgenic lines, restored the phenotypic aberrations of fugu5 pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis (pck1-2) or the glyoxylate cycle (icl-2; mls-2) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of fugu5 epidermal phenotype. fugu5 had oblong cotyledons similar to those of angustifolia-1 (an-1), whose leaf pavement cells display an abnormal arrangement of cortical microtubules (MTs). To gain insight into the genetic interaction between ANGUSTIFOLIA and H+-PPase in pavement cell differentiation, an-1 fugu5-1 was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, an-1 fugu5-1 pavement cells showed a striking three-dimensional growth phenotype on both abaxial and adaxial sides of cotyledons, which was recovered by hydrolysis of PPi in an-1 fugu5-1 AVP1pro::IPP1. Live imaging revealed that cortical MTs exhibited a reduced velocity, were slightly fragmented and sparse in the above lines compared to the WT. Consistently, addition of PPi in vitro led to a dose-dependent delay of tubulin polymerization, thus supporting a link between PPi and MT dynamics. Moreover, mathematical simulation of three-dimensional growth based on cotyledon proximo-distal and medio-lateral phenotypic quantification implicated restricted cotyledon expansion along the medio-lateral axis in the crinkled surface of an-1 fugu5-1. Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.

Loss of vacuolar acidity results in iron-sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis

BackgroundAutosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. MethodsWe generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. ResultsWe demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. ConclusionOur work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.

Expression of V-PPase proton pump, singly or in combination with a NHX1 transporter, in transgenic tobacco improves copper tolerance and accumulation.

One of the most important strategies evolved by plants to tolerate heavy metals (HMs) is their sequestration into the vacuole. Recent studies have demonstrated that Cu sequestration into vacuole is dependent on the electrochemical gradient generated by vacuolar proton pumps: the V-H+-PPase and the V-H+-ATPase. In a previous study, we demonstrated that co-expression of V-H+-PPase and a sodium/proton antiporter genes, isolated from wheat, in transgenic tobacco plants significantly increases both H+ pumping activity of the endogenous V-H+-ATPase and V-H+-PPase compared to wild-type (WT) plants, all grown in the absence of stress. In the present study, we evaluated the effect of expression, in tobacco, of vacuolar proton pump, TaVP1, singly or in combination with sodium/proton antiporter, TaNHXS1, on copper (Cu) tolerance and accumulation. Results showed that, when subjected to Cu stress, TaVP1 single transgenic tobacco lines exhibited a more robust root system, greater biomass production, less chlorophyll loss, lower MDA and H2O2 production, and higher catalase activity and accumulated more Cu than did WT. Interestingly, double transgenic tobacco lines exhibited the best Cu tolerance and accumulation than either of the single TaVP1 transgenic lines or WT plants, when subjected to excess Cu. In fact, double transgenic lines accumulated 2.5-fold and 1.9-fold more Cu than did WT and single TaVP1 lines, respectively. Thus, these results clearly demonstrate the usefulness of expression of vacuolar proton pump alone or in combination with sodium/proton antiporter as novel strategy for Cu phytoremediation.

TCIRG1 Transgenic Rescue of Osteoclast Function Using Induced Pluripotent Stem Cells Derived from Patients with Infantile Malignant Autosomal Recessive Osteopetrosis.

Osteoclasts are hematopoietic stem cell-derived multinucleated cells necessary for bone remodeling and resorption. TCIRG1 encodes a protein that is an adenosine triphosphate (ATP)-dependent vacuolar proton pump required for this process. Recessive loss-of-function mutations in both copies of this gene lead to impairment of osteoclast function, with increased bone density, increased skeletal mass, and early mortality. We isolated fibroblasts from a patient with the compound heterozygous TCIRG1 mutations c.1549G>A (p.517D>N) and c.2236C>T (p.746Q>X), and reprogrammed them into iPS (induced pluripotent stem) cells. The function of osteoclasts derived from these cells was then rescued by transgenic expression of TCIRG1 cDNA. In addition to the known effects of TCIRG1 loss of function, iPS cell-derived osteoclasts from this patient had reduced expression of the bone remodeling enzymes cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP), leading to reduced in vitro bone remodeling. Expression of both genes and pit formation were restored in iPS cell-derived osteoclasts following transgenic restoration of TCIRG1 expression. Transgenic overexpression of TCIRG1 was sufficient to restore osteoclast function in iPS cell-derived osteoclasts from a patient with infantile malignant autosomal-recessive osteopetrosis. This work provides a proof of concept for an autologous approach to treating osteopetrosis, potentially avoiding the risks associated with hematopoietic stem cell transplantation in a young patient population.

Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide.

During vinification Saccharomyces cerevisiae cells are frequently exposed to high concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth of unwanted bacteria or fungi present in the must. Up to now the characterization of the molecular mechanisms by which S. cerevisiae responds and tolerates SO2 was focused on the role of the sulfite efflux pump Ssu1 and investigation on the involvement of other players has been scarce, especially at a genome-wide level. In this work, we uncovered the essential role of the poorly characterized transcription factor Com2 in tolerance and response of S. cerevisiae to stress induced by SO2 at the enologically relevant pH of 3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO2, a percentage much higher than the one that could be attributed to any other stress-responsive transcription factor. Large-scale phenotyping of the yeast haploid mutant collection led to the identification of 50 Com2-targets contributing to the protection against SO2 including all the genes that compose the sulfate reduction pathway (MET3, MET14, MET16, MET5, MET10) and the majority of the genes required for biosynthesis of lysine (LYS2, LYS21, LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3, ARG7, ARG8, ORT1 and CPA1). Other uncovered determinants of resistance to SO2 (not under the control of Com2) included genes required for function and assembly of the vacuolar proton pump and enzymes of the antioxidant defense, consistent with the observed cytosolic and mitochondrial accumulation of reactive oxygen species in SO2-stressed yeast cells.