Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.
Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.
- MeSH
- Bacterial Proteins chemistry genetics isolation & purification metabolism MeSH
- Cell Membrane metabolism MeSH
- Enzyme Assays instrumentation methods MeSH
- Fluorescent Dyes chemistry MeSH
- Microscopy, Fluorescence instrumentation methods MeSH
- Mutagenesis MeSH
- Protozoan Proteins chemistry genetics isolation & purification metabolism MeSH
- Pyrophosphatases chemistry genetics isolation & purification metabolism MeSH
- Drug Design MeSH
- Recombinant Proteins chemistry genetics isolation & purification metabolism MeSH
- Fluorescence Resonance Energy Transfer instrumentation methods MeSH
- Saccharomyces cerevisiae MeSH
- Sequence Alignment MeSH
- Molecular Dynamics Simulation * MeSH
- Software MeSH
- Single Molecule Imaging instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.
- MeSH
- Amoxicillin metabolism pharmacology MeSH
- Anti-Bacterial Agents metabolism pharmacology MeSH
- Genes, Bacterial genetics MeSH
- Drug Resistance, Bacterial genetics MeSH
- Adaptation, Physiological genetics MeSH
- Helicobacter pylori drug effects genetics growth & development MeSH
- Microbial Sensitivity Tests MeSH
- Microbial Viability drug effects MeSH
- Pyrophosphatases genetics MeSH
- Gene Expression Regulation, Bacterial drug effects physiology MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.
- MeSH
- Biopsy MeSH
- Charcot-Marie-Tooth Disease blood genetics therapy MeSH
- Adult MeSH
- Phosphoric Diester Hydrolases genetics MeSH
- Transcription, Genetic genetics MeSH
- Genetic Markers genetics MeSH
- Glutathione Transferase genetics MeSH
- Glycoproteins genetics MeSH
- Cathepsin A genetics MeSH
- Skin pathology MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA, Messenger genetics MeSH
- Neuregulin-1 genetics MeSH
- PPAR gamma genetics MeSH
- Prognosis MeSH
- Disease Progression * MeSH
- Pyrophosphatases genetics MeSH
- Aged MeSH
- Treatment Outcome * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
OBJECTIVE: To identify the etiology of a novel, heritable encephalopathy in a small group of patients. METHODS: Magnetic resonance imaging (MRI) pattern analysis was used to select patients with the same pattern. Homozygosity mapping and whole exome sequencing (WES) were performed to find the causal gene mutations. RESULTS: Seven patients from 4 families (2 consanguineous) were identified with a similar MRI pattern characterized by T2 signal abnormalities and diffusion restriction in the posterior limb of the internal capsule, often also optic radiation, brainstem tracts, and cerebellar white matter, in combination with delayed myelination and progressive brain atrophy. Patients presented with early infantile onset encephalopathy characterized by progressive microcephaly, seizures, variable cardiac defects, and early death. Metabolic testing was unrevealing. Single nucleotide polymorphism array revealed 1 overlapping homozygous region on chromosome 20 in the consanguineous families. In all patients, WES subsequently revealed recessive predicted loss of function mutations in ITPA, encoding inosine triphosphate pyrophosphatase (ITPase). ITPase activity in patients' erythrocytes and fibroblasts was severely reduced. INTERPRETATION: Until now ITPA variants have only been associated with adverse reactions to specific drugs. This is the first report associating ITPA mutations with a human disorder. ITPase is important in purine metabolism because it removes noncanonical nucleotides from the cellular nucleotide pool. Toxicity of accumulated noncanonical nucleotides, leading to neuronal apoptosis and interference with proteins normally using adenosine triphosphate/guanosine triphosphate, probably explains the disease. This study confirms that combining MRI pattern recognition to define small, homogeneous patient groups with WES is a powerful approach for providing a fast diagnosis in patients with an unclassified genetic encephalopathy.
- MeSH
- Genes, Recessive genetics MeSH
- Infant MeSH
- Humans MeSH
- Mutation genetics MeSH
- Brain Diseases diagnosis genetics MeSH
- Child, Preschool MeSH
- Pyrophosphatases chemistry genetics MeSH
- Protein Structure, Secondary MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We assessed association between novel biomarkers of cardiovascular disease and conventional factors in 40 years old subjects (208 men and 266 women) from the general population of Slovakia. FERHDL (cholesterol esterification rate in HDL plasma), AIP – Atherogenic Index of Plasma [Log(TG/HDL-C)] as markers of lipoprotein particle size, and CILP2, FTO and MLXIPL polymorphisms, were examined in relation to biomarkers and conventional risk factors. Univariate analyses confirmed correlation between AIP, FERHDL and the most of measured parameters. Relations between AIP and CILP2, FTO and MLXIPL were not significant. However, CILP2 was significantly related to FERHDL in both genders. In multivariate analysis BMI was the strongest correlate of AIP levels. In a model where AIP was omitted, TG levels explained 43 % of the FERHDL variability in men, while in women HDL-C was the major determinant (42 %). In conclusions, FERHDL and AIP related to the known markers of cardiovascular risk provide means to express their subtle interactions by one number. Our novel finding of association between CILP2 polymorphism and FERHDL supports its role in lipid metabolism.
- MeSH
- Adult MeSH
- Esterification MeSH
- Extracellular Matrix Proteins genetics MeSH
- Genetic Predisposition to Disease epidemiology genetics MeSH
- Cholesterol, HDL blood MeSH
- Risk Assessment MeSH
- Polymorphism, Single Nucleotide genetics MeSH
- Humans MeSH
- Coronary Artery Disease epidemiology genetics blood MeSH
- Prevalence MeSH
- Pyrophosphatases genetics MeSH
- Risk Factors MeSH
- Statistics as Topic MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Slovakia MeSH
