A5079981662- Mitochondrial Function and Pathology
- DNA Repair Mechanisms
- Metabolism and Genetic Disorders
- RNA modifications and cancer
- HIV Research and Treatment
- Cancer-related Molecular Pathways
- ATP Synthase and ATPases Research
- Immunotherapy and Immune Responses
- interferon and immune responses
- Cytomegalovirus and herpesvirus research
- Angiogenesis and VEGF in Cancer
- Cancer, Hypoxia, and Metabolism
- Genetics and Neurodevelopmental Disorders
- RNA and protein synthesis mechanisms
- Anesthesia and Neurotoxicity Research
- RNA Research and Splicing
- RNA Interference and Gene Delivery
- Cancer Genomics and Diagnostics
- Folate and B Vitamins Research
- Advanced biosensing and bioanalysis techniques
- Carcinogens and Genotoxicity Assessment
- Metal-Catalyzed Oxygenation Mechanisms
- Chemotherapy-induced cardiotoxicity and mitigation
- CRISPR and Genetic Engineering
- PI3K/AKT/mTOR signaling in cancer
University of Padua
2008-2022
National Institutes of Health
2020
Yale University
2020
Harvard University
2020
Rockefeller University
2020
Cold Spring Harbor Laboratory
2018
Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots ubiquitous in organs. Here we identify general function regulation dNTP pools cultured cells. nuclear variably expressed during cell...
Eukaryotic cells contain a delicate balance of minute amounts the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for few minutes DNA replication. Both deficiency and surplus single dNTP may result in increased mutation rates, faulty repair or mitochondrial depletion. dNTPs are usually quantified by an enzymatic assay which incorporation radioactive dATP (or dTTP dATP) into specific synthetic oligonucleotides polymerase is proportional to concentration unknown dNTP. We find...
Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication repair. The mammalian enzyme consists of a catalytic (R1) radical-generating (R2 or p53R2) subunit. During S-phase, R1/R2 complex is the major provider deoxynucleotides. p53R2 induced by p53 after damage was proposed to supply repair translocating from cytosol cell nucleus. Similarly R1 R2 were claimed move nucleus during S-phase provide replication. These models suggest translocation...
In postmitotic mammalian cells, protein p53R2 substitutes for R2 as a subunit of ribonucleotide reductase. human patients with mutations in RRM2B , the gene p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from patient lethal homozygous missense mutation grow normally culture an unchanged complement mtDNA. During active growth, four dNTP pools do not differ size normal controls, whereas during quiescence,...
Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear are synthesized de novo from ribonucleotides by salvage thymidine in the cytosol. Mitochondria contain specific kinases deoxyribonucleosides that may provide compartmentalized synthesis Here we investigate source intra-mt phosphates their relationship to cytosolic pools isotope-flow experiments...
Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or salvage of deoxynucleosides. In cycling cells the pathway dominates, but quiescent becomes important. Two forms active mammalian reductases are known. Each form contains catalytic R1 protein, two differ with respect to second protein (R2 p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from cells. The recently discovered p53-inducible p53R2 was proposed be linked DNA repair...
We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of (mt) dNTPs as little 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159–12164). In cycling concentrations phosphates thymidine, deoxycytidine, deoxyadenosine (combining mono-, di-, each case) did not differ significantly between...
Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency cytosolic thymidine phosphorylase that degrades and deoxyuridine. The extracellular fluid patients contains 10–20 μm deoxynucleosides leading to changes in dTTP may disturb mtDNA replication. In earlier work, we suggested mt originates two distinct pathways: (i) reduction ribonucleotides cytosol (in...
Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase their plasma contains high concentrations deoxyuridine; elevation the corresponding triphosphates probably impairs normal mtDNA replication repair. To study metabolic events leading to MNGIE we used as model systems skin lung fibroblasts cultured in presence and/or...
Doxorubicin (Dox) is an effective antineoplastic drug with serious cardiotoxic side effects that persist after withdrawal and can lead to heart failure. Dysregulation of vascular endothelium has been linked the development Dox-induced cardiotoxicity, but it unclear whether how transient exposure Dox leads long-term downregulation Endothelial Vascular Growth Factor Receptor type2 (VEGFR2), essential for endothelial cells function. Using in vitro model devised study long-lasting brief Dox, we...
SAMHD1 is the major catabolic enzyme regulating intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates at Thr-592. How this modification affects function highly debated. We investigated role endogenous phosphorylation during cell cycle. Thr-592 occurs first G1/S border and removed mitotic exit parallel with Thr-phosphorylations most CDK1 targets. Differential sensitivity to phosphatase inhibitor okadaic acid suggested different involvement PP1...
The efficiency of Nucleotide Excision Repair (NER)process is crucial for maintaining genomic integrity because in many organisms, including humans, it represents the only system able to repair a wide range DNA damage. aim work was investigate whether photoproducts induced by UV-light affected circadian phase at which irradiation occurred. NER activity has been analyzed human quiescent fibroblasts (in absence cell cycle effect), rhythmicity synchronized with pulse dexamethasone. Our results...
Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 R2. A third protein, p53R2, with same function as R2, occurs in minute amounts. quiescent cells, p53R2 replaces absent humans, genetic inactivation of causes early death mtDNA depletion, especially muscle. We found that fibroblasts from a patient lethal mutation contained normal amount showed growth,...
Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence a 5′-nucleotidase (dNT2) with pronounced specificity dUMP and dTMP suggested that enzyme protects excess dTTP. In humans, accumulation dTTP causes genetic disease. now establish dNT2 in vivo indeed is located mitochondria. The native shows same substrate affinity inhibitors as...
In mammalian cells, the catabolic activity of dNTP triphosphohydrolase SAMHD1 sets balance and concentration four dNTPs. Deficiency leads to unequally increased pools marked imbalance. Imbalanced increase mutation frequency in cancer but it is not known if SAMHD1-induced imbalance favors accumulation somatic mutations non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated react constitutive pool characterized by a huge...
SAMHD1 was discovered in the immune system where it restricts HIV‐1 infection. Mutations lead to mitochondrial DNA deletions Aicardi‐Goutièrez syndrome. The protein is a deoxynucleoside triphosphate triphosphohydrolase. We study participation of catabolic enzymes regulation precursors (dNTPs) cultured mammalian cells. found that and its mRNA are present various cell types, including skin fibroblasts. Downregulation by siRNA transfection increases all 4 dNTP pools changes their relative...